P f'J' ~ ~ L.J (('{v • q?./'-(O

• OMAN FISHERIES DEVELOPMENT AND MANAGEMENT PROJECT

• . SEAFOOD QUALITY CONTROL LABORATORY MANUAL •

Contract Number 272-0106-C-00-I00I-00 • AID Project Number PlOtT 272-0106-3-0005

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PREPARED BY: • STEPHEN KELLEHER

PREPARED FOR: • CHEMONICS INTERNATIONAL CONSULTING DIVISION

SUBMITTED TO:

OMANI-AMERICA~~JOINT COMMISSION • FOR ECONOMIC & TECHNICAL COOPERATION • • TABLE OF CONTENTS

Preparation of reagents 1 • Sampling schedule 3 Treatment of data 5

Lipid determination - cold solvent extradition 6

Lipid determination - Soxhlet 8 • Lipid determination - acid hydrolysis 10 Protein - Kjeldahl 11

Non-protein nitrogen 14 • Protein - Biuret 15 Protein - modified Lowry 16

Moisture 17

Ash 18

• Carbohydrate 19

Trichloroacetic acid extract 20

Trimethylamine (TMA) 21 • Dimethylamine (DMA) 23 Thiobarbituric acid-reactive substances (TBARS) 25

Hydrogen ion concentration (pH) 27 • Element analysis 28 Cold vapor analysis 31

Methyl mercury 32

Histamine 34

• Bisulfites in shrimp 36

Fractionation of lipids 38

Fatty acid methyl esters 39 • Peroxide value 41 Conjugate dienes 43

Aerobic plate count 44 • • Salmonella 45

Staphlococcus aureus 46

Listeria monocytogenes 48 • Escherichia coli 49 Appendix •

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• • Seafood Quality Control Laboratory Manual • PREPARATION OF REAGENTS

Molar solution:

The preparation ofMolar solutions ofcompounds requires the compounds molecular • weight. This is best obtained from the label on the compounds container. Molecular weights obtained by adding up the molecular weights ofindividual elements may be satisfactory ifno other compounds are associated with the desired molecule. The most common compound to associate with a molecule is water (hydrated), which has to be accounted for in preparing reagents. Another common association compound is HeI. To aid in preparing a molar solution, the following examples are given: • Prepare I liter (1000 ml) ofa 3 M NaOH solution: The molecular weight ofNaOH is 39.98, therefore (39.98 g/mole) (3 moles/liter) = 119.94 g/liter

Prepare 500 ml ofa 3 M NaOH solution:

(39.98 glmole) (3 moles/liter) (500 ml/ 1000 ml ) = 59.97 g/500 ml

• Prepare 250 ml of a 3 mM NaOH solution:

(39.98 glmole) (0.003 moles/liter) (250 ml/lOOO ml) = 0.030 g/250 ml

(39.98 mg/mmole) (3 mmoles/liter) (250 ml/l000 ml) =30.0 mg/250 ml

• Prepare I liter ofa 0.5 M FeCI3 (anhydrous) solution: (162.20 glmole) (0.5 moles/liter) = 81.10 g/liter

Prepare I liter of a 0.5 M FeCI3 • 6 H20 solution: • (270.14 glmole) (0.5 moles/liter) = 135.07 g liter Ifa solution contains 21.04 g ofNaOH/500 ml, what is its molarity (M) ?

(21.04 g) / (39.98 glmole) = 0.53 moles/500 ml = 1.05 moles/liter = 1.05 M • DIMENSIONAL ANALYSIS Dimensional analysis is an examination ofthe units with the exclusion ofthe real numbers. This provides an excellent way to check the preparation ofreagents. For example:

(g) / (glmole) = mole (glmole) ( moles/liter)= g /liter • (g/mole) (moles/liter) (liters/ml)(ml/dl) = gldl Once the correct units are obtained, one can plug in the real numbers.

(21.04 g) / (39.98 g/mole) = 0.53 mole (39.98 glmole) (0.5 moles/liter) = 19.99 glliter (39.98 glmole) (0.5 moles/liter) (0.001 liters/ml) (100 mlldl) = 2.00 gldl • • Seafood Quality Control Laboratory Manual • LABORATORY REAGENTS FOR GENERAL USE (Source: CRC Handbook ofPhysics and Chemistry)

3 M Acetic acid: Dilute 172 rol ofconcentrated acetic acid (17.4 M, 99-100%) to 1 liter with distilled water. 3 M HydrocWoric acid (HCl): Dilute 258 m1 ofconcentrated HCl (11.6 M, 36%) to 1 liter with • distilled water. 3 M Nitric acid (HN03): Dilute 195 m1 ofconcentrated HN03 (15.4 M, 69%) to 1 liter with distilled water. 3 M Phosphoric acid (H3P04): Dilute 205 m1 ofconcentrated H3P04 (14.6 M, 85%) to 1 liter with distilled water. 3 M Sulfuric acid (H2S04): Dilute 168 m1 ofconcentrated H2S04 (17.8 M, 95%) to 1 liter with distilled water. • 3 M Ammonium hydroxide: Dilute 200 m1 ofconcentrated ammonium hydroxide (14.8 M, 28% NH;,) to 1 liter with distilled water. Aqua regia: Mix 1 part concentrated nitric acid (HN03) with 3 parts concentrated hydrocWoric acid (HCI). The formula should contain 1 part water ifthe aqua regia is to be stored for any length oftime. •

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• • 2 Seafood Quality Control Laboratory Manual • SAMPLING SCHEDULE

Regulation on Quality Controlfor Omani Fishery Exports Regulationfor Criteria & Standards ofFish Export April 4,1995 Association ofAnalytical Chemists, 1990, 15th ed., page 864.

• INTRODUCTION: Sampling ofproduct is critical to the success of a quality control program. Sampling takes place at two levels when monitoring products for quality. The first is batch to batch sampling where statistical formulas are adhered to, providing an established level offood safety while not destroying too much product in the process. For quality control ofOmani fisheries products, the batch sampling techniques as described in the "Regulation for Criteria & Standards ofFish Export" will be followed. The second level ofsampling is done with the individual product. Variations in chemical compounds contained in the fish can vary considerably throughout different anatomical regions ofthe • fish. Two techniques can be followed to assure consistent results. The first is to designate a specific anatomical area ofthe fish to be used for a chemical procedure. This does have some drawbacks in trying to exactly locate a specific area, but this technique is very useful in the analysis oflarge fish. With large fish AOAC suggests from each of~ 3 fish cut 3 cross sectional slices 2.5 cm thick, I slice from just back ofthe pectoral fins, I halfway between the first slice and vent, and 1 slice just back of vent. The second technique is to utilize the entire muscle, or component ofinterest, ofthe fish. AOAC suggests the use ofbetween 5-10 fish. All the muscle from the selected fish are ground up and mixed to • form a homogeneous paste. Sample aliquots are taken from this paste mixture. This technique is preferable to the first technique and works very well with small to medium size fish.

REGULATION FOR CRITERIA & STANDARDS OF FISH EXPORT:

General • • Fish shall be marked so as to identify each batch submitted. • Rejected or unsampled fish shall not be mixed with fish that have been sampled and accepted. • The required number ofsample units comprising the sample shall be drawn at random from the batch by an authorized officer for comparison with the appropriate standard. • Samples offish other than bulk loaded tuna, shall be assessed in accordance with Sampling Plan #1. • Where a batch is rejected on the basis ofan assessment under Sample Plan #1, the batch may be • reassessed immediately with a different sample in accordance with Sampling Plan #2. Ifthis is done, the initial rejection will not be counted unless the fish is again rejected. • Bulk loaded tuna shall be assessed in accordance with Sampling Plan #3.

Acceptance •A batch is considered as meeting requirements ofthe appropriate standard when the number of defective units does not exceed the acceptance number ofthe relevant sampling plan and all other • requirements ofthese Standards are complied with. •A batch will be rejected when: a) the acceptance number is exceeded; or b) any characteristic or condition ofthe fish that has rendered or may render the fish unfit for human consumption is detected. •A rejected batch may be eligible for rectification only ifthe number ofdefective units does not exceed 30 % ofthe number ofsamples taken. • •A resubmitted batch shall be reassessed in accordance with Sampling Plan #1. • Any sample unit found defective during inspection shall be rejected whether the batch as a whole is passed or rejected for export. • • 3 Seafood Quality Control Laboratory Manual • Sampling Plan #1

2-15 2 0 • 2-200 16-50 2 0 5-2400 201-1200 51-300 3 0 2401-12000 1201-7200 301-1400 6 1 12001-24000 7201-15000 1401-3500 13 2 24001-48000 15001-24000 3501-7200 21 3 48001-84000 24001-42000 7201-15000 29 4 84001-144000 42001-72000 15001-24000 48 6 •• 144001-240000 72001-120000 24001-42000 84 9 over 240000 over 120000 over 42000 126 13

Sampling Plan #2 •

2-15 3 0 2-200 16-50 5 1 5-2400 201-1200 51-300 6 1 2401-12000 1201-7200 301-1400 13 2 • 12001-24000 7201-15000 1401-3500 21 3 24001-48000 15001-24000 3501-7200 29 4 48001-84000 24001-42000 7201-15000 48 6 84001-144000 42001-72000 15001-24000 84 9 144001-240000 72001-120000 24001-42000 126 13 over 240000 over 120000 over 42000 200 19 • Sampling Plan #3

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The level ofacceptance for each chemical component is determined by the country to which to the fisheries product is being exported. Since these values are constantly under review, it would be • advisable to check the levels ofacceptance just prior to shipment.

• • 4 Seafood Quality Control Laboratory Manual • TREATMENT OF DATA

In reporting data there are certain fonnulas that are common place. The following is a compilation of • some ofthe basic concepts needed to report data. Mean:

Mean = ~ all samples / n the number of sample • Example: Values = 2,5,7,9 The mean = ~ 2,5,7,9/4 = 23/4 =5.75 Standard deviation:

(j = (L ( x - Mean)2 / number ofsample) y, • Example: Average = (2 + 3 + 4)/3= «2-3i + (3-3)2 + (4-3i / 3) v, = 3.0 ± 0.82 Propagation ofError

Attached to scientific values is a number (±), which gives an indication as to the uncertainties associated with that number. In many cases it is the standard deviation ofthe data used to obtain that number. When manipulating groups ofnumbers, all with associated uncertainties, there are rules for • their treatment. Addition and Subtraction:

• Example: (23 ± 4) + (16 ± 6) + (12 ± 8) = 51 ± 10.8 2 2 2 (4 + 6 + 8 ) y, = (116) v, = 10.8

Multiplication and Division: • Example: 23 ± 4 / 3 ± 2 = 7.67 ± 0.69

«4 /23i + ( 2/3i) v, = (0.47)V, = 0.69 •

• • 5 Seafood Quality Control Laboratory Manual • LIPID DETERMINATION - COLD SOLVENT EXTRACTION

Bligh, E. G. and Dyer, WJ. 1959 Can. J. Biochem. Physiol. J2911. Palmer, F.B. St.. c. 1971 Biochim. Biophys. Acta 231:134.

• INTRODUCTION: The procedure ofBligh and Dyer is the most commonly used procedurefor the extraction oflipid from seafood products. Its use of a three, then two phase system allows for the extraction ofboth neutral and membrane lipids assuring an extraction oftotal lipid. The use oflow temperatures throughout the extraction process provides a practically unmodified lipid, suitable for further analytical analysis (oxidation state, fatty acid methyl esters (FAMES, etc.). • REAGENTS: Chloroform

Methanol ( dried with anhydrous sodium sulfate ifnecessary).

0.88% Potassium Chloride (KCI): Add 0.88 g KCl to a 100 ml volumetric flask. Bring to • volume with distilled, deionized water PROCEDURE:

• Place a representative 50 g sample offish muscle into a stainless steel chalice blender containing 50 ml chloroform and 100 ml dried methanol. • • Blend four times on high speed, 30 seconds each, with intermittent ice chilling ofthe blender flask.

• Add 50 ml chloroform and blend for an additional 30 seconds.

• Add 50 ml of0.88% potassium chloride and blend for 30 seconds. (Sample should develop a • slightly white appearance) • Filter homogenate through Whatman #1 filter paper in a Buchner funnel under slight vacuum.

• The residue and filter paper are re-blended with 100 ml offresh chloroform and re-filtered.

• Rinse blender and residue with an additional 50 ml chloroform.

• • Combine filtrates and transfer to a separatory funnel and allow to stand until complete separation and clarification.

• Remove bottom chloroform layer, filter, and deposit into a tared round bottom flask. • • Vacuum (Flash) evaporate (35 - 40°C) offsolvent until dry. • Purge flask containing lipid with nitrogen or argon for 2 minutes and cap.

• Place round bottom flask into a dessicator until cool and dry.

• Re-weigh round bottom flask to calculate percentage lipid. • • 6 Seafood Quality Control Laboratory Manual • CALCULATIONS:

% LPIPID = (wgt. of flask after evaporation) - (wgt. of flask empty) X (100 /wgt. ofsample)

• Note: The weights ofthe sample andsolvents in this procedure can vary as long as the ratios are consistent with the originalprocedure. It is imperative that the volumes ofchloroform: methanol: water before and after dilution, be kept at 1:2:0.8 and 2:2:1.8 respectively. The procedure described above assumes that the fish sample is 80% moisture. Ifsamples vary considerablyfrom this moisture content, then either adjustment in sample weight or volume ofsolvents used will have to be changed to reflect • that the proper amount ofwater is present.

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• • 7 Seafood Quality Control Laboratory Manual • LIPID DETERMINATION - SOXHLET

Application Note Soxtec System HT6 Manual (I'ecator Hoganiis. Sweden)

INTRODUCTION: The extraction oflipid using the Soxhlet procedure is widely used for grains, • seeds, flours and other non-animal products, where a majority ofthe lipid is ofneutral type. The use of cWoroformlmethanol does allow the procedure to extract both membrane and neutral lipid and hence works well for the extraction oflipid from seafood. The use ofhigh temperatures in this procedure radically modifies the lipid, thus disallowing its use in extracting lipid for subsequent analysis. • REAGENTS: Appropriate Solvent: diethyl ether 100°C recommended temperature petroleum ether 110°C " hexane 140 °C " " chloroform/methanol (2:1) 160°C " "

• PROCEDURE:

Service Unit:

• Check level ofService Unit to assure silicon oil is at the correct level. The volume ofoil must be enough to lift up the floating switch. Never fill higher than 3 cm below the tank lid, to allow for • expansion and to avoid splashing . • Turn on the unit by pushing the "POWER" button located on the front ofthe unit. To set/reset new temperature, depress "READ/SET" switch on the unit's front panel until the display reads "Set". Adjust the new desired temperature by turning the "SET" knob until the display reads the desired temperature. Re-press the "READ/SET" button until the "SET" lamp goes out. The display now reads the actual temperature ofthe silicon oil. When the temperature has reached the set • temperature (-2%) the "READY" lamp goes on.

Extraction:

• Open the cold water to cool the condensers. Flow should be adjusted to approximately 2 • liters/minute to prevent solvent evaporation. • Thoroughly grind fish tissue or fish meal until particle size of 1 mm or less. Fish tissue may be ground with the aid ofsand or Celite ifneeded.

• Attach thimbles to the thimble adapter rings using gloves to prevent oils from hands from • contaminating the thimbles. • Place approximately 3 grams (accurately weighed) into separate Soxtec thimbles. For weighing, use the spare thimble support tared to O.OOOg. All transfer ofthimbles should be performed using the thimble handler. Thimble is released when handle is depressed.

• Cover sample with a thin layer ofdry lipid-free cotton wool, and place the thimbles and adapter rings containing sample into the thimble supports attached to their holder.

• • Insert the thimbles into the condenser, raising the holder up into the condensers. The unit should be in the "RINSING" position. • • Move each knob into the boiling position to allow the magnet to attach to the adapter ring. 8 Seafood Quality Control Laboratory Manual •

• Re-Iower the knob to the "RINSING" position, which allows the thimble to hang below the condenser; then remove the thimble supports.

• Tare the metal cups (Boiling chips are included in weight ifused).

• • Insert the six extraction cups filled with 25-50 m1 ofsolvent.

• Place the cups under the condensers and move the knob to the "BOILING" position, which will place the thimbles directly into the solvent. Push left handle down until a tight seal is achieved above cups. • • Be assured that the condenser tubes are open and boil sample and thimbles for recommended time. (Approximately 40 minutes).

• Move the knobs to the "RINSING" position and continue solvent flow for 5 minutes.

• After "rinsing" is complete, close condenser valves to allow solvent to collect in the condensers.

• • When almost all ofthe solvent is retained in the condensers, press the "AIR" button on the service unit and open the "EVAPORATION" valve (pushed up) on the extraction unit until all traces of solvent are removed from the thimbles. (Approximately 10 minutes).

• Close the "EVAPORATION" valve and remove the empty cups. • • Place the thimble supports under the thimbles and lower the handle, letting the thimbles drop into the supports.

• Release handle, following the upward movement with support holder while removing thimbles.

• Additional samples can be performed by reusing the solvent in the condensers; add new cups • (without solvent) and thimbles and repeat process. • When complete, turn offthe "POWER" switch on the Service Unit and stop cold water to condensers; remove solvent using safety. Solvent should be disposed ofin a manner compatible with hazardous waste. • Place cups in a 100°C oven for approximately 1 hour, then cool in a glass desiccator. • • Re-weigh cups to determine weight gain due to crude lipid.

CALCULATIONS: • % LIPID = weight ofcups (after) -- weight ofcups (before) I weight ofsample

• • 9 Seafood Quality Control Laboratory Manual • LIPID DETERMINATION - ACID HYDROLYSIS METHOD

AOAC 15th ed Method 964.12 Rapid Modified Babcock Method, page 871. First Action 1964 • ( Applicable to raw, canned, andfrozen fish) INTRODUCTION: The appeal for this procedure is due to the fact that it is rapid and easy to perform. The Babcock method is widely used in the dairy industry, and modifications to the digestion bottle have allowed for its use with fish, cheese and meat products. The digesting agent acid cleaves the lipid free from any associations and allows it to coalesce above the aqueous phase, where it is read directly in a calibrated tube. Due to the harsh acid environment, the extracted lipid is radically •• modified and not suitable for subsequent analysis.

REAGENTS:

Acetic acid, glacial

Trichloroacetic acid, 70% (note Trichloroacetic acid was substitutedfor perchloric • acid due to perchloric hazards.). Dissolve 70 g oftrichloroacetic acid into 30 m1 distilled water.

Digesting reagent: Acetic acid! Trichloroacetic acid (50/50) (vol./vol.).

• PROCEDURE: • Weigh 9.0 g. ground and mixed fish tissue into a Paley-type Babcock cheese bottle. (Kimble Glass, No.508, 20% size).

• Stopper and add approximately 30 m1 digesting reagent.

• • Heat for approximately 20 min., while stirring in a water bath maintained at 92± 2 cC.

• Remove from heat and add more digesting reagent until fat layer is in calibrated neck ofthe bottle.

• Centrifuge for 2 min. at approximately 600 RPM in a table top centrifuge. • • Iflipid falls below calibration, add more digesting reagent, centrifuge 1 minute, then read again. • Read percentage fat directly from calibrated area using the bottom ofthe top meniscus.

• With fish high in lipid, it may be necessary to use less than 9 g sample. In this case, correct • reading by multiplying lipid by the factor 9 / g sample.

• • 10 Seafood Quality Control Laboratory Manual • PROTEIN· KJELDAHL

Kjeltec Auto 1030 Analyzer Manual (Tecator, Hoganiis, Sweden)

INTRODUCTION: The original procedure was developed in 1883 by a Danish • scientist and has been modified numerous times throughout time. It is the most frequently used procedure for the measurement ofprotein for almost every type ofcommodity. The procedure utilizes sulfuric acid, which hydrolyzes and converts all the protein nitrogen to the stable compound ammonium sulfate. Under alkaline conditions ammonia gas is liberated from the ammonium sulfate and trapped in boric acid, which becomes neutralized. The degree ofneutralization ofthe boric acid, which can be determined by titration with acid, is ultimately dependent on the amount ofnitrogen in the original sample. Once the percent nitrogen is determined, a factor is used as the multiplier to convert nitrogen • to protein. Since nitrogen represents 16% ofthe total weight ofthe average protein, then 1/0.16 or 6.25 is used many times as the conversion factor. It is the factor most commonly used for seafood.

REAGENTS:

Concentrated Sulfuric Acid (H2S04)

• 0.20N HCI: Place 66.667 ml of 3.00 M HCI (see Preparation ofReagents) into a 1000 ml volumetric flask that contains pproxiametely 700 ml distilled water. Swirl and bring to volume with additional distilled water.

Catalyst: For each digestion tube add 0.1 g copper sulfate and 5.0 g potassium sulfate, alternative is the purchase ofKjeltab C.

• Sodium Hydroxide 40%: In a fume hood place 400 g ofNaOH pellets into a beaker and add approximately 500 ml distilled water. Stir solution, and let cool to room temperature. Place solution into a 1000 ml volumetric flask and bring to volume with distilled water.

Sodium Hydroxide 0.1 M: Place 4.0 g NaOH pellets into a 1000 ml volumetric flask. Bring • to volume with distilled water. Receiving solution: Dissolve 100 g boric acid in 10 liters ofdistilled water. Add 100 ml bromocresol green solution ( 100 mg bromocresol green in 100 ml methanol), prior to the addition of70 ml methyl red solution (100 mg methyl red dye - alcohol soluble, in 100 ml methanol). Transfer 25 ml to an Erlenmayer flask and add 100 ml distilled water. Ifsolution is red titrate with 0.1 M NaOH until neutral gray color is obtained. Calculate the amount of NaOH needed to adjust the 10 liters ofreceiving solution according to the formula: • mil.O M NaOH =ml titer x 40 Add the calculated amount of NaOH to the remaining 10 liters ofreceiving soution. Alternative is to purchase Kjelsorb ready-made receiving solution.

Steam generator water: Add 0.5 g NaCI to each tank offresh distilled water to act as an electrolyte and improve conductivity.

• DIGESTION:

• Tum on digester in hood at the highest setting, (425°C) to warm up (approximately 30 min).

• Label tubes for identification

• Add between 1 - 2 g (accurately weighed) well mixed sample into separate tubes (samples can be • weighed on nitrogen-free, ashless paper, andsample andpaper added to the tube). • Add 2 Kjeltab C tablets (or catalyst) to each tube. • 11 Seafood Quality Control Laboratory Manual • • Dispense 12 ml concentrated sulfuric acid (H2S04) into each tube.

• Place the glass venting apparatus onto the tubes and turn on aspirator water to create vacuum.

• Place the tubes into the digester block and digest for 40 min.

• • After completion ofdigesting remove tubes and stand and allow to cool.

• Add 75 ml distilled water to each tube. • DISTILLAnON: • Check and top offall liquid in tanks, and that drain valve to steam generator is in the closed position.

• Switch on "POWER" • • Press "REC.SOL." button 4-5 times to flush out the system. • Open the cold tap water to a flow ofabout 2 liters per min.

• Insert an empty tube and close the safety door.

• Press "STEAM" button on (upwards) and let steam flow into tube for approximately 1 min. • (Ifthe water temperature is too hot the unit will automatically shut off. Increasing the cooling. waterflow will reduce the temperature ofthe thermo switch, which will automatically turn on the steam after the proper temperature has been reached.

• Press "STEAM" to offand the instrument is now ready for use.

• • Select the Kjeldahl program by pushing the switch in the upward direction.

• Adjust the constants to read "A"= 00.00, "B"= 1.000, and the blank to 0.000

• Open the safety door and press "AUTOIRESET" to "AUTO".

• Place the tube with blank onto the rubber fitting and close the safety door. ( A blank is made by • digesting 12 ml concentrated H S0 and 2 Kjeltabs without sample). 2 4

• Record the value for the blank and dial it into the "BLANK" setting on the front panel ofthe distillation unit.(As the blank number is dialed into "BLANK" the display reading decreases in unison) • • With the new blank value set, readjust the "B" value to 1.751 (This adjusts the instrument to display % protein directly if0.2 N Hel is used as the titrant and 6.25 the conversionfactor).

• Place sample tube with unknown protein content into distiller and shut safety door.

• Sample is automatically run. • • 12 Seafood Quality Control Laboratory Manual • CALCULATIONS:

Values of% Protein are presented in display which when divided by sample weight gives actual % Protein. (In the determination ofproteinfrom seafood using the Kjeldahl procedure, a non-protein • nitrogen analysis should always be performed).

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• • 13 Seafood Quality Control Laboratory Manual • NON-PROTEIN NITROGEN

Woyewoda, A.D. et al. 1986 Recommended Laboratory Methodsfor Assessment ofFish Quality, Can. Tech. Report ofFish. andAquatic Sci. No 1448, Dept. ofFish. and Oceans, P.o.Box 550, Halifax, • Nova Scotia, B3J2S7 INTRODUCTION: The use ofthe Kjeldahl procedure for the measurement ofprotein is widely used for many commodities. It measures nitrogen content in products, which when multiplied by a factor (t), represents protein concentration. In most products this works quite well, but in seafood products large amounts ofnitrogen, not associated with protein, cause interference. These non-protein nitrogen sources are in the form ofamines (trimethylamine and its oxide, dimethylamine, free amino acids) urea, nucleotides and purine based compounds. With the use oftrichloroacetic acid, protein is precipitated, • leaving non-protein nitrogen sources in the supernatant or non-precipitated layer. This layer ofnon­ protein nitrogen is examined by the Kjeldahl procedure, and its results are subtracted from the protein content obtained in the original sample.

REAGENTS: • Trichloroacetic acid (10%): Dissolve 100 g trichloroacetic acid in 900 ml distilled water. PROCEDURE:

• Homogenize 50 g fish muscle in 100 ml of 10% trichloroacetic acid (cold) in a Waring blender on high speed for 1 minute. • Let homogenate set for 5 minutes on ice. • • Re-blend on high speed for an additional 30 seconds. • Filter homogenate through Whatman #1 filter paper in a Buchner funnel under slight vacuum. • Place between 1.0 g to 2.0 g (accurately weighed) offiltrate into aKjeldahl tube and proceed with the Kjeldahl procedure as previously described on page ,to determine non-protein nitrogen.

CALCULATIONS: • Values of percent Non-protein are presented in the Kjeltec display, which when divided by the sample weight (l-2g), and multiplied by 3, gives actual % Non-protein.

%PROTEIN -% NON-PROTEIN = % ACTUAL PROTEIN •

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• • 14 Seafood Quality Control Laboratory Manual • PROTEIN - BIURET

Layne, E. 1957. In: Methods in Enzymology, Vol.3, p. 450, Academic Press, Inc. New York Torten, J and Whitaker, JR. 1964 J Food Sci. 29:168-174.

• INTRODUCTION: Protein measured by Biuret reaction methods are used quite often in biochemistry laboratories. They are simple and rapid to perform on almost all types ofsamples. The sodium hydroxide in the reagent produces a highly alkaline environment, which completelysolubilizes the proteins. In this environment the peptide bond, which is a characteristic ofproteins, reduces copper from the +2, to the +1 state. This reduced state copper then reacts with the sodium, potassiumtartrate to produce a chromophore with a maximal absorbance around 540 nm. Since it is actually a measure of peptide bonds, no interference is obtained from non-protein nitrogen or free amino acids. It is therefore • thought to be a direct and accurate measure oftotal protein.

REAGENTS:

Biuret Reagent: Dissolve 1.50 g CUS04 • 5 H20 and 6.0 g ofNaK.C4~06 • 4 H20 in 500 ml distilled water. Add with swirling 300 ml of !O% NaOH ( 100 g NaOH in 1000 ml volumetric flask with distilled water). Cool and bring to 1000 ml volume with distilled • water. Store in a polyethylene bottle for up to 6 months. Ifa black precipitate should appear, discard solution.

Sodium Deoxycholate 10%: Dissolve 10 g ofsodium or potassium deoxycholate into 90 ml ofdistilled water. • PROCEDURE: • Place 1.0 ml ofassay fluid, adjusted to contain protein within a readable range, in a test tube. • Add 4.0 ml ofBiuret Reagent and immediately mix on a VorteX® mixer on high speed. • Let stand at room temperature for at least 30 minutes. (Color is stable up to at least 2 hours). • Read absorbance ofsolution at 540 nm. • Ifcloudiness appears in the cuvet and interferes with the absorbance readings, add 0.01 to 0.03 ml • of 10% deoxycholate solution. Iflarger volumes are used they must be accounted for in the dilution factor. (Sanae Mii, University ofWisconsin, Madison, WI).

CALCULATIONS:

A standard curve is constructed using Bovine Serum Albumin (BSA) in concentrations ranging from • 0.5 mg/ml to 10 mg/ml.

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• • 15 • Seafood Quality Control Laboratory Manual PROTEIN - MODIFIED LOWRY

Markwell, UK., Haas, S.U, Bieber,L.L and Tolbert, NE. 1978 Analytical Biochem. 87:206 Lowry, OH et al. 1951 J. BioI, Chem. 193:265-275.

• INTRODUCTION: The chemistry behind this procedure is similar to the Biuret reaction, where copper reduced by the protein's peptide bond reacts with sodiumtartrate to produce a chromaphore. In the Lowry procedure the addition of Folin-Ciocalteu Phenol reagent increase the sensitivity ofthe procedure 100 times more than the Biuret procedure, capable ofreaching into the Ilglml range. The addition ofthe SDS acts to solubilize membranes (lipid), thus releasing membrane bound protein for quantitative detection. '. REAGENTS: Reagent A: 2% NazC03, 0.4% NaOH, 0.16% sodium tartrate and 1% sodium dodecyl sulfate (SDS).

Reagent B: 4% CUS04 • 5 H20 • (Both solutions can store indefinitely when stored at room temperature)

Folin-Ciocalteu Phenol Reagent

Bovine Serum Albumin (BSA) • PROCEDURE:

• Mix 100 parts Reagent A and 1 part Reagent B to form Reagent C, the alkaline copper reagent. • In a test tube, place 1 ml ofsample that contains protein within a readable range. • Add 3 ml Reagent C and incubate at room temperature for 30 minutes. • Add 0.3 ml ofdiluted ( 1:1) Folin -Ciocalteu Phenol Reagent and mix on a Vortex® mixer. • • Incubate an additional 45 minutes at room temperature. • Measure absorbance at 660 nm using an identically prepared sample, minus the protein, as the control blank.

CALCULATIONS: • A standard curve is constructed using BSA ranging from 10 - 100 Ilg protein / tube.

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• • 16 • Seafood Quality Control Laboratory Manual MOISTURE

Woyewoda, A.D. et al. 1986 Recommended Laboratory Methods for Assessment ofFish Quality, Can. Tech. Report ofFish. andAquatic Sci. No. 1448, Dept. ofFish. and Oceans, P. o Box 550, Halifax, Nova Scotia, B3J2S7

• INTRODUCTION: Moisture content is a measure ofthe amount ofwater contained in a sample. Water can be bound to the chemical components in the seafood either loosely (free) or tightly. Loosely bound water is driven offfIrst when the sample is placed into an oven. As loosely held water becomes rapidly depleted, the rate at which total water is removed from the sample decreases substantially. A slow rate ofwater removal then occurs until no additional water can be removed from the sample. On larger sample, water is removed rapidly from the surface, at which time a crusting ofthe surface occurs called "case hardening". This dry surface then acts as a barrier to water escape and traps water inside • the sample leading to artifIcially low moisture readings. To prevent this samples should be uniform and spread r cut as thin as possible.

PROCEDURE:

• Place empty aluminum weighing dishes into a pre-heated oven heated to 10J'C for a minimum of2 hours, or until a constant weight has been obtained. • • Place empty weighing dishes into a dessicator to cool, prior to weighing to the nearest 0.0001 g. • Place approximately 10 g (accurately weighed) into each dish. • Place the samples into the 103°C oven and remain there overnight. • Place dried samples into a dessicator to cool, and re-weigh samples to 0.0001 g. (Samples high in lipid should be dried and weighed as quickly as possible, due to the fact that lipid will take up • oxygen and increase in weight over time). CALCULATIONS:

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• • 17 • Seafood Quality Control Laboratory Manual ASH

Windsor, M andBarlow, S. 1981 Introduction to Fishery By-Products, Fishing News Books Ltd., Farnham, England, pages 150-151.

INTRODUCTION: Ash is a measure ofthe total inorganic component ofa sample, which is made up • substantially ofminerals. Two methods ofashing are commonly used: 1) wet ashing in which organic compound are degraded by digesting samples in an oxidizing acid such as nitric orperchloric, or by the use ofnon-oxidizing acids, such as hydrochloric or sulfuric, followed by an oxidizing agent, usually hydrogen peroxide, or 2) dry ashing, which takes place in a furnace at temperatures ranging between 400°C to 700°C. Dry ashing is the most common method used to determine total mineral content of seafood.

• PROCEDURE:

• Place porcelain crucibles and covers into a muffle furnace, which has been pre-heated to 550'C, for a minimum oftwo hours. • Remove the empty crucibles and covers and place them into a dessicator until cool. • Weigh to the nearest 0.0001 g. • • Place approximately 4 g (accurately weighed) ofpreviously dried sample (105 'C oven, overnight) into each cru(:ible and properly place on its cover. • Place the crucibles into the furnace at 5500C and ash overnight (finished ash samples should be white andfree ofblack carbon particles). • Re-weigh crucibles and covers to 0.0001 g. • CALCULATIONS: (wgt. crucible, cover, ash - wgt. crucible, cover) ASH CONTENT = (wgt. crucible, cover, sample - wgt. crucible, cover) X 100 •

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• • 18 Seafood Quality Control Laboratory Manual • CARBOHYDRATE

INTRODUCTION: Carbohydrates are the components composed of saccharides ofvarying lengths and chemical composition. In foods for human consumption these are loosely grouped into sugars and starches. There are methods for the detection oftotal carbohydrates, such as the Phenol-Sulfuric acid • method, but proximate analysis determines carbohydrate by using the following equation: %Carbohydrate = 100-(% protein + % lipid + % moisture + % ash)

In seafood, the carbohydrate content is usually very low or barely detectable. Therefore, ifa large concentration ofcarbohydrate is determined from the formula above, the accuracy ofthe other • component determinations should be checked.

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• • 19 Seafood Quality Control Laboratory Manual • TRICHLOROACETIC ACID EXTRACT

INTRODUCTION: Trichloroacetic and perchloric acids are the two most commonly used protein precipitants. In recent years perchloric has been used less due to its explosive nature when dehydrated. Trichloroacetic acid levels between 5-10% are found to be most effective, because it is in this range that • the acid changes the protein's electrical charges (+,-) to be approximately equal in number. This is known as the isoelectric range and is where the protein is most susceptible to precipitation. Theory states the reason as lack ofrepulsive forces caused by like charged proteins being at a minimum. Like charged molecules repel each other and increase solubilization ofthe molecules. Also, the abundance ofopposite charges attract to each other and cause coagulation followed by precipitation.

• REAGENTS:

10% trichloroacetic acid: Dissolve 100 g trichloroacetic acid in 900 ml distilled water.

PROCEDURE:

• • Homogenize 50 g fish muscle in 100 ml of 10% trichloroacetic acid (cold) in a Waring blender on high speed for I minute. • Let homogenate set for 5 minutes on ice. • Re-blend on high speed for an additional 30 seconds. • Filter homogenate through Whatman #I filter paper in a Buchner funnel under slight vacuum. • (Extracts are stablefor at least 6 months atfrozen (_40 0 C) temperatures).

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• • 20 Seafood Quality Control Laboratory Manual • TRIMETHYLAMINE (TMA)

Dyer, w.J. and Mounsey, Y.A. 1945 J. Fish. Res. Bd. Canada 6:359. Tozawa, H et al. In: Inspection and Quality Control. Fishing News (Books) Ltd., London. p 187.

• INTRODUCTION: Many species ofsea-life contain trimethylamine oxide (TMAO) as an osmoregulator or anti-freeze compound. During iced or chilled storage, TMAO gets reduced to trimethylamine (TMA) by bacterial enzymes. TMA is odoriferous contributing to the amine or ammonia odor ofaged fish. TMA has also been used quite extensively as a chemical indicator of chilled fish. Its correlation to sensory evaluation is not always good, and fluctuations have been found due to differences in species, initial concentrations ofits precursor TMAO, and catch seasons. In spite ofthese problems TMA is still frequently, used and the following picric acid method has been accepted • by the Association ofAnalytical Chemists (AOAC).

REAGENTS:

Ttrimethylamine standard:

stock: Dissolve 0.6820 g ofTMA-HCI in distilled water, and dilute to 100 • ml in a volumetric flask with distilled water. Store refrigerated.

working: Dilute 1 ml ofstock TMA solution to 100 ml with distilled water.

Formaldehyde (10%): Dilute 26.8 ml of37.3% fonnaldehyde solution (fonnalin) to 100 ml with distilled water.

• Potassium hydroxide solution (25%): Dissolve 25 g ofcrystalline potassium hydroxide (KOH) into 75 ml distilled water. Cool before use.

Picric acid solution:

stock: Place 2 g dried (in a dessicator overnight, DO NOT DRYINAN OVEN!) in • a 100 ml volumetric flask and dilute to volume with toluene (dried). working: Dilute 1 ml ofstock picric acid solution to 100 ml with toluene (dried).

Toluene • Sodium sulfate (anhydrous) PROCEDURE:

• Prepare trichloroacetic acid extract as described in preceding procedure. • Pipet between 0.1 - 4.0 ml aliquots (depending on degree ofspoilage) into a screw top tube. Add distilled water to each tube to bring total volume to 4.0 ml. • • Add 1.0 ml of fonnaldehyde (10%0 to each tube. • Place 10 ml ofdried toluene into each tube. • Pipet 3.0 ml ofpotassium hydroxide (25%) solution into each tube. • Mix on a rotator or other stirring device at 30°C for 15 minutes. • Remove between 6 - 8 ml of toluene (upper) layer with apasteur pipet and place into a test tube that contains approximately 0.4 g sodium sulfate (anhydrous). After drying ( approximately 5 minutes) place 5.0 ml ofthis solution into a clean screw top test tube. • • Pipet 5.0 ml ofworking picric acid solution into sample containing screw top tube. • Cap and swirl tube gently, prior to measuring its absorbance at 410nm in a glass cuvet. • 21 • Seafood Quality Control Laboratory Manual CALCULATIONS:

• Prepare a standard curve using 1.0, 1.5,2.0, and 3.0 rol ofworking standard solution. This is equivalent to 100, 150,200, and 300 Ilg TMA-N respectively. • Add distilled water to each tube to bring the volume to 4.0 rol. • • Add formaldehyde, toluene, and potassium hydroxide as described in above procedure. • Plot Absorbance at 410 nm (Y axis) versus Ilg TMA-N (X axis).

TMA-N STANDARD CURVE

• 0.5 -,------, •

0.4 • E 0.3 o" • • ~ • g 0.2 -em • ] « 0.1

• 0.0

o 50 100 150 200 250 300 350 ug TMA-N

• Absorbance / 0.0013 =Ilg TMA-N (Ilg TMA-N /1000) (75) (4/ ml sample) =mg % TMA-N •

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• • 22 Seafood Quality Control Laboratory Manual • DIMETHYLAMINE (DMA)

Dyer, w.J. and Mounsey, YA. 1945 J.Fish. Res. Ed. Can. Q(J):359-367.

• INTRODUCTION: In the frozen state trimethylamine oxide (TMAO) undergoes an enzymic transformation, utilizing TMAO-ase, to become dimethylamine (DMA) and formaldehyde, in equimolar concentrations. Formaldehyde has been suggested to playa major role in the toughening offlesh texture in gadoid species offish, hence decreasing quality. Formaldehyde is very difficult to quantitatively determine due to its high reactivity. Therefore, DMA is used as a·measure of formaldehyde production and therefore overall quality. The difference between moles ofDMA and moles ofactual formaldehyde, is used as an indicator ofthe amount offormaldehyde that has reacted • with the fish tissue.

REAGENTS:

Dimethylamine standard:

stock: Place 116.5 mg DMA - HCl into a 100 ml volumetric • flask. Add 1 ml concentrated HCl (with caution!) , and bring to volume with distilled water.

working: Dilute 1 ml DMA stock standard to 100 ml with distilled water in a volumetric flask.

• Copper - ammonia reagent: Dissolve 20 g ammonium acetate and 0.2 g CUS04 • 5 H20 41 30 ml distilled water. Dissolve 109 sodium hydroxide in 25 ml distilled water, and wait until it cools, prior to the addition of20 ml ammonium hydroxide. Add the 30 ml ammonium acetate solution to the hydroxide solution. Bring up to 100 ml volume in a volumetric flask with distilled water. Transfer to a polyethylene bottle.

Carbon disulfide (5%): Dilute 25 ml ofcarbon disulfide to 500 ml with benzene (When • handling benzene, gloves andworking in a hooded area are minimum precautions). Acetic acid (30%): Add 30 ml glacial acetic acid to approximately 50 ml distilled water. Bring up to 100 ml volume with distilled water. • Sodium sulfate (anhydrous) PROCEDURES:

• Place an aliquot ( 1 -2 ml ) neutralized trichloroacetic acid extract in a 25 ml screw top test tube.

• Dilute to 10 ml with distilled water.

• • Add 1.0 ml copper - ammonia reagent and mix.

• Add 12.0 ml ofcarbon disulfide (5%) solution in benzene.

• Heat loosely stoppered tubes for 5 minutes in a 37° - 40°C water bath. • • Tighten caps. Shake for 5 minutes (manual, or orbital shaker). • Immediately add 1.0 ml of30% acetic acid, re-cap, and shake by hand until top layer is clear (about 30 - 40 times). • 23 Seafood Quality Control Laboratory Manual • • Decant benzene layer into a test tube containing approximately 0.4 g ofanhydrous sodium sulfate. Shake gently.

• Decant dried benzene solution into a glass, spectrophotometer cuvet and measure its absorbance at 440 nm. A blank should be constructed as above substitutingtricWoroacetic acid for • tricWoroacetic acid extract.

CALCULATIONS:

Absorbance ofsample Neutral vol. vol ofextract 100 Absorbance per mg DMA X Unneutral vol. X vol ofaliquot X wgt. of sample

• = mg DMA-N 1100 g fish

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• • 24 Seafood Quality Control Laboratory Manual • 2-THIOBARBITURIC ACID REACTIVE SUBSTANCES (TBARS)

Lemon, D. W 1975 An Improved TBA Test For Rancidity, New Series Or. No. 51, Halifax Laboratory, Halifax, Nova Scotia, Canada

• INTRODUCTION: There are two basic methodologies to measuring TBARS. One extracts the lipid from the fish sample prior to chemical evaluation, while the other is called the whole muscle method. This procedure is ofthe later type. As lipid in the seafood sample oxidizes, it produces higher concentrations ofa di-aldehyde named malonaldehyde. In the presence ofthiobarbituric acid, malonaldehyde forms a chromaphore with maximal absorbance around 530nm. Monitoring TBARS, especially for frozen product, has been shown to be an effective chemical evaluation tool for monitoring the degree oflipid oxidation or overall quality in seafood. Recent investigations have • shown that TBARS values are produced from a variety ofcompounds undergoing oxidation, such as proteins, and carbohydrates, and that components oflipid other thanmalonaldehyde contribute to increased TBAR values. Many investigators have shown excellent correlation between TBARS and sensory scores, which explains why it is one ofthe most widely used chemical tests to monitor oxidation. • REAGENTS: Extracting Solution: In a 1000 ml volumetric flask, add 75 g trichloroacetic acid, 1.0 g n-propyl gallate, and 1.0 g ethyleneaminetetraacetic acid (EDTA). Bring up to volume with distilled, deionized water.

TBA Reagent: In a 1000 ml volumetric flask, add 2.338 g of2-thiobarbituric acid and bring to volume with water. Slight warming may be necessary tosolubilize • particulates.

Both above solutions should be kept refrigerated in dark colored, polyethylene bottles. They are both stable for a minimum of6 months. • PROCEDURES: • Blend 15 g tissue in 30 ml cold extracting solution for 30 seconds in a Waring blender, or equivalent, on highest speed. (The amount ofsample to solution can vary, but the ratio ofthe two must stay at 1:2, sample:solution).

• Filter the homogenate through Whatman #1 filter paper, placed in a funnel.

• • Mix 5.0 ml offiltrate with 5.0 ml ofTBA reagent in a screw cap tube. Filtrate may be diluted with distilled water (quantitatively), ifextensive oxidation is suspected. ( The amount offiltrate and TBA reagent may vary, but must always be in a 1:1 ratio).

• Heat in boiling water bath for 20 minutes. • • Cool and measure absorbance at 530 nm.

CALCULATIONS:

Quantitative evaluation ofTBARS is determined through the use ofa standard curve prepared using 1,1,3,3, tetraethoxypropane (YEP) in concentrations ranging from 0 to O.051J1ll01e. After use YEP • should be flushed with an inert gas (nitrogen, or argon) and stored in a refrigerator. (Absorbance - 0.0425/11.1402) (600) = ~moles TBARSI kg fish tissue. • 25 Seafood Quality Control Laboratory Manual • TBARS STANDARD CURVE

0.7

0.6 • • 0.5 • •

E c 0.4 • 0 <") l.l) Q) c0 0.3 • • -eIII • 0 (f) .0 0.2 « • 0.1 • 0.0

0.00 0.01 0.02 0.03 0.04 0.05 0.06 umoleTBA •

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• • 26 Seafood Quality Control Laboratory Manual • HYDROGEN ION CONCENTRATION (pH)

INTRODUCTION: The pH ofan aqueous solution is a measure ofits hydrogen ion concentration. The fonnula pH =log l/(H) = -log (H). It is also a measure ofa sample's acidity level. A pH 00.0 is considered neutral, < 7.0 is acidic, and> 7.0 is basic or alkaline. The pH is not a measure oftotal • acidity, which has to be determined by another method, usually titration. The pH measurement can not be used for total acidity due to buffering effects caused by chemical components in the sample, such as proteins or phosphate groups It is however, an extremely useful measurement that gives volumes of infonnation about the stability, both chemically and microbiologically, and its reactivity with other compounds. It is perhaps, the most important infonnation one can obtain from a chemical test about a seafood product.

• REAGENTS: Distilled water

Standardized bnffers: pH 4.0, pH 7.0 • PROCEDURES: • Calibrate pH meter according to individual instrument directions with standardized buffers at pH 4.0 and 7.0. • Homogenize fish muscle in room temperature distilled water (1:10 w/v) for 30 seconds. • Place pH probe into mixture and read pH directly. •

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• • 27 Seafood Quality Control Laboratory Manual • ELEMENT ANALYSIS

INTRODUCTION: The use ofAtomic absorption spectroscopy is routinely used for the analysis of most elements, such as copper, iron, and lead to name a few. Acid digested samples are aspirated up into a flame which causes the elements in the sample to gain energy and push their electrons from • ground state to ones ofhigher, more unstable energy. Since many elements are "atomized" at the same time a mechanism ofpassing a selected wavelength through the flame during atomization allows the instrument to differentiate between elements; the wavelengths used are specific for each element and are supplied by hallow cathode or electron discharge lamps. A few elements are more efficiently determined using vapor analysis, either cold or slightly warm. These elements are mercury, and those that fonn hydrides, such as arsenic and selenium. In these procedures areductant, usually sodium borohydride, is added to a sample to produce an element vapor along with hydrogen, which propels the • vapor into a quartz cell in line with the wavelength beam. The wavelength, again, is specific for each element. In both techniques, standard curves are produced using pure elements at varying concentrations, to produce quantitative assessments ofthe elements.

REAGENTS:

Diluting solution: To a 1000 ml volumetric flask containing 400 ml distilled water, add 58 • ml ofconcentrated nitric acid and 67 ml ofconcentrated sulfuric acid. Bring to volume with distilled water.

Nitric acid solution (1 %): Place approximately 500 ml ofdistilled water into a 1000 nil volumetric flask. Carefully add 109 ofconcentrated nitric acid prior to bringing to volume with additional distilled water.

• Element standards: Dilute the purchased element standard (1000 ppm) with a 1% nitric acid solution until the desired range is obtained. _

PROCEDURE: • All glassware and utensils used in handling product should be acid washed and rinsed with polished distilled water. (Read: Parr Microwave AcidDigestion Bomb Operational Instruction Manual before using the apparatus for the first time!)

Digestion in a Parr Microwave Acid Digestion Bomb: • • Place 0.1 g ofrepresentative homogeneous sample into a Parr bomb Teflon sample holder that has been acid washed and distilled water rinsed. • Carefully add 2.5 ml ofconcentrated (70%) nitric acid to the sample. • Screw down the top ofthe bomb until fully closed, then hand tighten 1/8th ofa turn more. Do not over tighten! Make sure bomb is dry ofmoisture on its outside surface. • Place bomb into a microwave and turn on high heat for 30-45 seconds. • Let digestion bomb cool for approximately 30 minutes before attempting to open. Cool water may • be used to speed up the process. • Open bomb and place contents into a 100 ml volumetric flask, and bring to volume with diluting solution. • Determine element content on the sample as described below. • ATOMIC ABSORPTION SPECTROMETER - PERKIN ELMER MODEL 3300 Alignment oflamps and name optimization:

• Turn on equipment in the order: 1. AA Spectrometer, 2. Printer, 3. Computer. (Let system • warm up for at least 20 minutes prior to use). 28 Seafood Quality Control Laboratory Manual

• • Turn on pressure regulator on the bottled gases and adjust the pressures as follows: Acetylene 15 psig., Zero air 50 psig.; (Air from a compressor will have no pressure gauge). • Type "GEM" into computer at the MS-DOS prompt (C:I» This command lets one enter the AAS "DESKTOP".. • Double click on the "AA_INST. EXE." icon. This command lets one enter the AAS "BENCHTOP". • • In the "BENCHTOP" mode the "3300" icon in the middle ofthe bottom ofthe screen changes from a dimmed blue to a more intense, darker blue, indicating that there is communication between the computer and the AAS. • Move pointer to the "WINDOWS" heading and click on "ALIGN LAMP". • Once in the "ALIGN LAMP" screen, with the mouse, click on desired lamp and element until both become highlighted. (Click on both even ifthey are already highlighted) (This causes the turret to move lamp into correct position).. • • Open up the plastic shield that covers the lamp turret. • On the lamp that is in position (facing operator in the upper position), adjust both turnscrews, one at a time, until the energy signal on the monitor (blue stripe) reaches its maximum. Stop. Click on the "AGC/AlC" icon, which re-scales the energy, and continue to turn screws until maximum is reached again. Continue doing this until the ultimate maximum is reached, and push 'AGC/AlC' once more. • Repeat the above alignment procedure pulling out or pushing in the lamp until a maximum signal is • obtained, click on "AGC/AlC". • Once alignment ofthe lamps has been accomplished, exit the "ALIGN LAMP" screen by clicking on the "43" symbol in the upper left region ofthe page. • Ifflame alignment is needed, go to the "WINDOWS" heading and click on "CONTINUOUS GRAPHICS". • The burner is aligned by the adjustment ofthree positions knobs: vertical, horizontal, and • rotational. • Open the flame compartment door and locate the vertical adjustment knob. • Rotate the knob until the burner head blocks the light beam as indicated by an increase in absorbance. • Rotate the knob until the absorbance is 0.000, then give the knob one-quarter ofa turn more in the same direction. • Do not exit the "CONTINUOUS GRAPHICS" screen, but under the "WINDOWS" heading click • on "FLAME CONTROL" heading to light the flame. • Once in "FLAME CONTROL" make sure that the "INTERLOCK" icon is not in the highlighted mode. (Ifit is, then one ofthe four safetyfeatures has been disarmed and must be remedied). • Assure that the proper conditions are displayed on the screen, such as air, acetylene. (The default flow rate for acetylenel air mixture should be 3.018.0, and light the flame by pushing the "FLAME" icon. (Ifthe flame does not light right away, it may help to bleed the gases for a few seconds and try again). • • Exit the "FLAME CONTROL" screen by clicking the "43" icon to return to the "CONTINUOUS GRAPHIC" screen. • Adjust both the "Horizontal" and "Rotational" knobs until the absorbance ofthe flame reaches a maximum value.

• Calibration and Element Concentration: • Under the "WINDOWS" heading, click on "ELEMENT PARAMETERS". (Before a sample can be run, an Element Parameterfile has to be created). • In the first screen "INST" the two-letter chemical symbol for the element should be entered, i.e.Pb, CU,As, etc. • The analyst should enter his/her name. • Signal type for most analyses should be "AA". • • Slit width should be pre-selected from the element input, but ifone desires to change it, click on the "_" symbol and select the desired slit width. The height should always be in the high mode for flame analysis. • 29 Seafood Quality Control Laboratory Manual • • Read time and delay can be altered ifnecessary. • Sample and standard replicates are chosen next, the default is 1. • Gas low qualities are selected in the last box on the page. • Click on the "CALIB" heading. • This sets up the calibration or standard curve. Enter under the "CONC", column across from S1, the concentration ofthe first standard. (Example: 5, which standsfor 5 mg/liter in the default • mode. Remember, mg/liter is the same as parts per million or ppm). • Fill in the remaining standards under S2, S3, etc. • The calibration equation should be left non-linear, and the units for both calibration and samples should read mgIL. • Click on the "OPTIONS" heading. • This is a printer options page and allows one to dictate what is to be printed in the report. (The • default supplies enough detailfor most reporting needs). • Save the element parameters by selecting the "FILE" menu and choosing "SAVB AS". • Enter the name ofthe file to be stored. (Example: PbTUNA). Click on "OK" to save. (This file can be selectedforfUture determinations by entering "ELEMENTPARAMETERS", then going to the "FILE" menu andselecting "OPEN". At the prompt enter the file which is to be run, andskip over the "INST", "CALIB", and "OPTIONS" settings. • Exit out of"ELEMENT PARAMETERS" but clicking on the "43" icon, returning to the • "BENCHTOP" screen. • Double click on the "MANUAL" icon. • Four windows are simultaneously displayed at once. Click anywhere on a window to bring it to the front ofthe screen. Only windows at the front ofthe screen are active and capable ofbeing acted upon. • Click on the "CALIBRATION" heading on the upper toolbar. • Place nebulizer sipper into the blank sample, wait approximately 5 seconds, then click on • "AUTOZERO". Rinse the sipper with clean, distilled water (between samples, as well)and continue to place standards, in the order that they are highlighted on the screen(SI then S2, etc.. ,). Click the correct sample after it has been sipped for a minimum of5 second& • To read the sample after the calibration curve has been completed, click anywhere on the "MANUAL CONTROL" screen. (One click may be needed on an empty space to hide the calibration menu prior to bringforward the "MANUAL CONTROL" screen with one more click). • • Place the nebulizer sipper into the sample and click on the "READ" icon. Continue to sample until the "READ" icon becomes un-highlighted. Rinse sipper with distilled water and continue to read all samples in a similar fashion. • At the conclusion ofall the sample readings, exit all four screens by clicking on the "43" icon on each ofthe four windows. • • This returns the program to the "BENCHTOP" screen. Shut down of Instrument:

• Turn From the "WINDOWS" heading choose the "FLAME CONTROL" screen. • Click on the "FLAME" icon to shut offthe flame. • Close the valve on the acetylene tank. • Click on the "BLEED GASES" icon until the acetylene gas pressure gauge reads zero. • • Close the valve on the air supply. • Click again on " BLEED GASES" until either the pressure gauge reads zero with bottled gas, or there is no more venting sound when using a compressor. • Turn offthe AAS, followed by the printer and finally the computer.

CALCULATIONS:

• ppm Element x 1000 = ppm Element/ g fish tissue • 30 Seafood Quality Control Laboratory Manual • COLD VAPOR ANALYSIS (MRS-I0, MERCURYIHYDRIDE SYSTEM)

INTRODUCTION: The use ofthe Mercury/Hydride apparatus on the atomic absorption spectrometer is very similar to the standard analysis just described with the following exceptions: 1) a quartz cell is used in place ofthe open air above the burner. • 2) with the quartz cell in place, the absorbance reading is always minimized, not maximized as it is with a flame. 3) from the "BENCH-TOP" screen the "MS-I0" icon is double clicked versus the "FLAME" icon. 4) In mercury analysis no flame is used, and the instrument should be cooled for approximately one-halfhour ifthe flame had recently been utilized. 5) the element is delivered to the spectrophotometer by the MS-l0 rather than by a '. nebulizer. REAGENTS:

Hydrocloric acid solution (1.5%): Place approximately 500 ml ofdistilled water in a 1000 ml volumetric flask. Carefully add 1.5 g ofconcentrated hydrochloric acid, and bring to • volume with additional distilled water. Sodium hydroxide solution (1%): Place 1 g ofsodium hydroxide pellets into a 100 ml volumetric flask and add approximately 900 ml ofdistilled water. Stir and let cool to room temperature. Bring to volume with additional distilled water.

Sodium borohydride solution (3%): To be made fresh prior to each analysis, add 3 g • sodium borohydride to a 100 ml volumetric flask. Bring to volume with 1% sodium hydroxide solution.

PROCEDURE:

• It is strongly suggested that one reads the MS-l0 User Documentation prior to using the apparatus • for the first time. Also, it is critical to make sure the venting hood above the spectrophotometer is on. This is more critical than when using flame analysis due to the evolution ofhydrogen gas during cold vapor analysis. . • Turn on the spectrometer and as before click the appropriate lamp for the element to be analyzed. A 20 - 30 minute warm-up period is recommended. • Digest the sample in a Parr Microwave Acid Digestion Bomb as described above. • Dilute the digested sample to 100 ml using 1.5% hydrochloric acid solution. • • Attach the MS-l0 apparatus to the spectrophotometer following the manual instructions. • Fill reservoir bottle (left side container) with 3% sodium borohydride solution. • Turn on argon gas at 36 psig pressure. • Connect an empty reaction flask (right side container) to the flange, wait for 10 seconds, then push down plunger on the MS-l0 and hold to the "READ" icon on the manual display screen dims. • Press autozero • • Disconnect the reaction flask and place 10 ml of 1.5% hydrochloric acid solution containing varying standards to construct a standard curve. Enter calibration curve standards as described above for the blank and also for regular element analysis. • Place 10 ml ofdiluted digested sample into the reaction flask and repeat process.

• When procedure has been completed, neutralize the sodium borohydride solution with weak acid and discard. Do not store.

• CALCULATIONS: ppm Element x 1000 =ppm Element! g fish tissue • 31 Seafood Quality Control Laboratory Manual • METHYL MERCURY

AOAC 16th ed. Method 988.11 Rapid Gas Chromatography Method, page 24

INTRODUCTION: Mercury has been fOlmd to accumulate in the lipid fraction ofsome species of • fish, mainly those ofthe Scombroid family. Many countries have put legal limits on the concentration ofmercury that can be allowed in the fish tissue. European nations uses the total mercury limit of Ippm / g oftissue. Methyl mercury represents a very large proportion oftotal mercury ill seafood and has been adopted by the United States as its mercury indicator compound. In this procedure fish sample is washed repeatedly with acetone to remove interfering substances, prior to its extraction with toluene. The extract is then analyzed by electron capture (ECD), which detects chlorinatedorganics (CHzHgCI). • REAGENTS: Hydrochloric acid solution (50%): Carefully add 50 ml concentrated hydrochloric acid to 50 ml distilled water

Acetone, Toluene, and Isopropanol

• Methyl mercuric chloride standards: stock: Dissolve 0.1252 g ofmercuric chloride in toluene to a total volume of 100 ml (volumetric). 1000J.l.g Hg/ml intermediate: Dilute 0.01 ml ofstock solution to 1.0 ml (volumetric) O.IJ.1.gHglml working: Dilute 0.1, 0.2, 0.4, 0.6, and 0.8 ml to 1.0 ml (volumetric) for a • concentration of 0.01, 0.02, 0.04, 0.06, and 0.08 J.l.g HglmI. PROCEDURE:

• Accurately weigh 1.0 g ofrepresentative fish tissue that has been ground and mixed until it is homogeneous. • Place sample into a 50 ml centrifuge tube and add 25 ml ofacetone. • • Shake vigorously by hand for 15 seconds. • Loosen cap and centrifuge for 5 minute on high speed in a table top centrifuge. • Discard acetone (Use a pipet ifnecessary). • Repeat acetone cleaning step two additional times. • Add 20 ml oftoluene, cap, and vigorously shake by hand for 30 seconds. • Loosen cap and centrifuge as before. • Discard toluene • • Add 25 ml ofhydrochloric acid solution to the washed sample, and shake gently. • Add 20 ml oftoluene and gently shake for 5 minutes by hand. • Loosen cap and centrifuge as before. • Ifemulsion is present, add 1 ml iso-propanol and gently stir with a glass rod. (Vigorous mixing may cause interfering peaks), prior to centrifugation. • Transfer, using a pasteur pipet, the toluene layer to a graduated cylinder. • • Wash walls ofcentrifuge tube with clean toluene (approximately 2 ml), and transfer add to solvent in graduated cylinder. • Extract with 20 ml toluene as before one more time, and add to solvent in graduated cylinder. • Bring volume ofextract to 50 ml using fresh toluene. • Add 109 anhydrous sodium sulfate and mix • Stopper container and store refrigerated for up to 15 hours, prior to analysis by gas chromatograph. • • 32 Seafood Quality Control Laboratory Manual • Gas Chromatograph Conditions:

Column: 6 ft. x 2mm (id.) glass, packed with 5% DEGS-PS on 100-120 mesh Supelcoport.(Condition column according to manufacturers directions or refer to AOAC method mentioned above for details), column temperature 155°C. Carrier gas: Argon or Nitrogen with oxygen scrubber and molecular sieve, 30 ml/minute. • Injector: 200°C Detector: 63Ni, electron capture, 300°C

CALCULATIONS:

Plot peak areas from standards (Y-axis) versus concentrations ofHg (X-axis) to obtain the straight-line • equation using the least square treatment ofdata. J.1g Hg / g fish =(X J.1g) (vol. sample inj./ vol. stnd inj.) (50)

(X J.1g) obtained from standard curve. •

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• • 33 Seafood Quality Control Laboratory Manual • mSTAMINE

AOAC 15th ed. Method 977.13 Fluorometric Method, page 876

INTRODUCTION: Histamine is in a class oforganic chemicals named biogenic amines. Others • included in this group include cadaverine and putrescine. All these amines are produced from the decarboXylation ofan amino acid. Histamine's precursor ishistadine. Public health concerns focus on histamine because it has been associated with scrombroid poisoning attributed mainly to tuna and fish from the mackerel family. Histamine is a vasoactive compound that has the ability to cause dilation of blood vessels, headaches, cramps, and nausea. It is for this reason that it is ofconcern to seafood exporters. Regulations on Quality Control for Omani Fishery Exports that fish from the Clupeidae, Scombridae, Scombresocidae, Pomatomidae, and Coryphaenedae families shall not contain histamine in • excess of 10 mg/IOO g based on the average ofsamples tested, or 20 mg/IOO g in any sample unit.

REAGENTS:

Methanol: Dried with anhydrous sodium sulfate ifneeded.

Sodium hydroxide (2M): Dissolve 80 g sodium hydroxide pellets into approximately 900 ml • distilled water. Stir and let cool prior to bringing to volume with distilled water.

Sodium hydroxide l.OM): Dissolve 40.00 g sodium hydroxide pellets into approximately 900 ml distilled water, let cool, and top offvolume with additional distilled water. . • Phosphoric acid (3.57 N): Dilute 121.8 ml of85% H3P04 to 1000 ml with distilled water. 0.1 % o-pthalidicarboxaldehyde (OPT): Dissolve 100 mg OPT in 100 ml fresh methanol. Store in an amber bottle in refrigerator. Prepare fresh weekly.

Hydrochloric acid (l.OOM): Dilute 33.33ml of3M HCI (see Preparation ofReagents) to 100 ml with distilled water in a volumetric flask. • Hydrochloric acid (O.IM): Dilute 3.33 ml of3M HCI to 100 ml with distilled water in a volumetric flask.

Histamine solution: (prepare all fresh weekly)

stock: ( I mg/ml): 169.1 mg into 100 ml (vol.), using O.IN HCl. intermediate: ( 10 Ilg/ml): 1 ml stock histamine into 100 ml (vol.), using • O.IN HCl. working: (0.5 -1.5 Il g/5 ml): 1,2, and 3 ml intermediate histamine into separate 100 ml (vol.), using 0.1 N HCl.

Ion Exchange Resin: (fresh weekly)

Add 15 ml 2N NaOH / g. resin; swirl let stand approx. 30 min., decant off • liquid. Repeat alkaline wash using equal volumes ofNaOH. Wash with water; filter and repeat water wash. Store resin in water.

PROCEDURE: • Methanol Extract: 10 g. offish tissue is homogenized in 50 ml methanol (cold) in a Waring blender at high speed for 2 min. Homogenate is transferred to a 100 ml • volumetric flask heated to 60°C for 15 min, then cooled to 25°C, before 34 Seafood Quality Control Laboratory Manual • being brought to volume with methanol. Filter liquid throughWhatman # 1 filter paper; store refrigerated for up to several weeks.

Column Separation:

Pipet 1 mI ofextract onto the colunm and add an additional 4-5 mI water. • Initiate colunm flow into 50 mI volumetric flask containing 5.00 mI of 1.00 N HCI. Elute with water until approximately 35 mI has accumulated in flask. Dilute to volume with water.

Fluoresces Reaction:

Pipet 5 mI eluent into 50 mI Erlenmeyer flask. Add 10 mI, 0.1 N HCI. Pipet • in 3 mI, IN NaOH and mix. Within 5 min pipet in 1 mI ofOPT solution and mix. After exactly 4 min, pipet in 3 mI of3.57 NH3P04 and mix. Within 1.5 hours read fluorescence at 350 nm excitation, and 444 nm emission.

CALCULATIONS: • A standard curve is developed using a histamine standard with concentrations of 0.5, 1.0, and 1.511g/5 mI. Plot fluorescence (Y axis) versus llg ofhistamine (X axis) and determine the straight line equation using the least square treatment ofdata.

mg. Histamine 1100g fish =(X llg 15 ml) (10) (Dilution factor). •

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• • 35 Seafood Quality Control Laboratory Manual • BISULFITES IN SHRIMP

AOAC 16th ed. Method 990.28 Optimized Monier-Williams Method, page 29

INTRODUCTION: The use ofa bisulfite dip for shrimp is common in the fishing industry. Upon • storage some shrimp will develop a defect termed "black spot". The use of bisulfites has been shown to be effective at reducing or eliminating this defect. However, a certain segment ofthe population is allergic to the sulfites and require safeguarding against this additive. A residual limit of100ppm 802 has been shown to be effective at minimizing black spot while posing little or no harm to the population. • REAGENTS: Hydrochloric acid solution: To 60 ml ofdistilled, deionized water, place 30 ml of concentrated HCI.

Methyl Red Indicator: Dissolve 250 mg methyl red in 100 ml ofethanol.

Sodium hydroxide (O.OlOM): Dissolve 03998 g NaOH pellets into approximately 900 ml • distilled water. Stir, cool and bring to 1000ml in volumetric flask with additional water. Certified reagent may also be purchased.

Hydrogen Peroxide (3%): For each analysis dilute 3 ml of30% hydrogen peroxide to 30 ml with distilled water. Just prior to use add 3 drops ofmethyl red indicator andtitrate with sodium hydroxide (0.01 OM) until yellow point. Ifend-point is exceeded, discard solution.

• Nitrogen: Use ultra high purity gas that has been filtered through an oxygen trap.

Ethanol (95%): Place 900 ml ofabsolute ethanol into a 1000ml volumetric flask, and bring to volume with distilled water.

• PROCEDURE: • Assemble Monier-Willams apparatus as described in the glassware's directions, or in the AOAC procedure listed above.

• Transfer 50 g ofa representative sample ofshelled and deveined shrimp to a Waring blender.

• • Add 100 ml ethanol (95%).

• Briefly blend the shrimp sample into course chunks (Only enough to allow sample passage through the neck ofthe ground glass round bottom flask) ..

• Add 400 ml distilled, deionized water to round bottom flask.

• • Close stop cock to prevent liquid passage from the separatory funnel to the round bottom flask, and add 90 ml hydrocWoric acid solution to separatory funnel.

• Tum on nitrogen flow at a rate of200 ± 10 ml/minute, and turn on cooling water for condensers.

• Add standardized hydrogen peroxide (3%) to the Monier - Williams receiving flask.

• • Remove separatory funnel and add sample (in ethanol) to the round bottom flask. • 36 Seafood Quality Control Laboratory Manual • • Open stop-cock ofseparatory funnel to dispense approximately 87 ml of4 N Hel (leave approximately 2-3 ml ofthe hydrochloric acid solution to prevent S02 escape through the separatoryfUnnel)

• Turn on power to heating mantle to a setting that allows liquid to exit the condenser at a rate of80­ • 90 drops a minute. • Boil contents for 1 hr and 40 min.

• Remove receiving vessel from the apparatus and titrate the SOzIHzOz solution until a yellow end­ point is reached, using sodium hydroxide(O.OlOM).

• CALCULATIONS:

SOl (ppm) =(32.03) (X ml NaOH) (0.010) (1000) wgt. ofsample •

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• • 37 Seafood Quality Control Laboratory Manual • FRACTIONATION OF LIPID INTO POLAR AND NEUTRAL COMPONENTS

Choudhury, R.B.R. andArnold. L.K. 1960 J. Am. Oil Chemists' Soc. Vol. 37:87-88.

INTRODUCTION: Lipid is divided into two main categories, non-polar and polar. The non-polar are • also termed neutral lipid and are the major constituent in depot or storage lipid. The lipids are extracted almost completely into single phase non-polar solvents, such as pentane, hexane,methylene chloride, or chloroform. Polar lipids are also called membrane lipids because this is where they"are predominantly located. Their polarity comes from a charged component on one ofthe fatty acids. The charged component in seafood membranes is almost exclusively a phosphate group. To extract polar lipids, a polar solvent, such as methanol or isopropanol is frequently used.

• REAGENTS:

Silicic acid: Heat in a dry oven for at least 2 hours at 125°C to activate on a Pyrex® tray.

Chloroform • Methanol: dried with anhydrous sodium sulfate ifnecessary. PROCEDURE:

• In a 125 ml Erlenmeyer flask, add 2 - 3 g. oflipid extracted using the method ofBligh & Dyer (1959), 25 g ofactivated silicic acid and 50 ml chloroform. • Place on a orbital shaker or manually shake for lO minutes. • • Pour total contents into a glass separation column, which has either a sintered glass filter or is plugged at the bottom end with glass wool. • Wash the contents ofthe column with five 50 ml aliquots offresh chloroform. • Combine filtrates and evaporate to dryness using a rotary evaporator. (This lipid represents the neutrallipidfraction). • Wash the column with five 50 ml aliquots offresh, dried methanol. • Combine filtrates and evaporate to dryness using a rotary evaporator. (This lipid represents the • neutrallipidfraction).

CALCULATIONS: • (wgt. of each fraction Iwgt. oftotal lipid sample) X 100 = % neutral or polar lipid.

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• • 38 Seafood Quality Control Laboratory Manual • FATTY ACID METHYL ESTERS (FAMES)

Shewfelt, RL. 1984 Ph. D. Thesis, University ofMassachusetts, Amherst, Massachusetts

INTRODUCTION: The analysis oflipid requires a post extraction chemical modification to allow • for its separation using gas chromatography. The most common chemical modification which produces individual component volatility is methylation. The incubation ofthe lipid with an acid chloride or fluoride in an alcohol environment, converts the triacylglycerol into fatty acids, and then converts the fatty acids into esters, compatible with the alcohol. Hence, fatty acids produced in ethanol will produce ethyl esters, propanol will produce propyl esters, and methanol will develop into methyl esters. Component volatility is essential to the separation ofcompounds using gas chromatography because the mobile phase is inert gas. Ifthe compound can not enter into this phase it will get "locked" onto the • column, only to be purged with very high temperatures. Separation ofFAMES allows one to examine the chemical make-up ofthe lipid. Retention times ofcompounds on a column is specific for that compound and can be used for identification purposes. Retention times are the same whether the compound is alone or as a complex mixture. Many fatty acids in seafood lipids are known for being very long, between 18 to 22 carbons in length. They are also noted for being highly unsaturated with as many as 6 double bonds. This high degree ofunsaturation makes them valuable for certain purposes, but also makes them highly susceptible to a spoilage called oxidation. Knowledge ofthe chemical • composition ofthe seafood lipids allows for the greater use ofthese lipids in industrial and food products.

REAGENTS:

14% Boron trifluoride in methanol (BF3): This is a standard chemical that comes • supplied in small (5 ml) one-use ampules. Petroleum ether: Dried with anhydrous sodium sulfate.

Internal standard: Place 109.2700 mg of C-23 (tricosanoic acid) fatty acid into a 100 m1 volumetric flask and bring to volume with petroleum ether. • PROCEDURE: • Place approximately 50 mg (accurately weighed) ofextracted lipid (Bligh & Dyer) into a medium size screw top test tube.

• Add 5 m1 ofinternal standard in petroleum ether into the screw top tube containing the lipid.

• • Remove solvent from the test tube by drying in a stream ofnitrogen gas.

• Add a 5 m1 ampule ofBF3/ methanol to the tube and tightly screw on test tube top.

• Place test tube into a boiling water bath for 60 minutes. • • Upon cooling, add 1.0 m1 distilled water and 2.0 ml fresh petroleum ether. • Mix thoroughly and centrifuge at medium speed in a table top centrifuge for 5 minutes.

• Remove the petroleum ether layer using a pasteur pipet and place into a clean test tube.

• Re-extract additional esters by adding 5 m1 fresh petroleum ether to the remaining liquid in the • original screw top test, then mixing and centrifuging again. • Repeat this extraction step on the sample one additional time (for a total of3) and combine all three petroleum ether extracts. • 39 Seafood Quality Control Laboratory Manual • • Evaporate offthe petroleum ether under a stream ofnitrogen in a 400C water bath until near dryness.

• Add fresh petroleum ether to bring the total volume to 2.0 mi. • • Inject 0.5 III onto a gas chromatograph column. Gas Chromatograph Conditions:

Column: Supelcowax-IO capillary column (polar bonded phase is silica) Carrier gas: Helium 30 psig Flame Ionization Detector (FID): 280°C, fueled with hydrogen and compressed air. • Injector: splitless, 250°C Temperature program: Initial: 160°C for 4 minutes Program rate: 4°C I minute Final: 225°C for 2 minutes

CALCULATIONS: • (peak area of unknown peak I peak area ofinternal standard) x 5.4635 =mg ofsample (To determine the order in which the FAMES exit the column, it is crucial that a multiple standard be purchased that contains many ofthe FAMES ofinterest. Supelco, Belafont, PA, sells a complete line of standards on an individual bases or as a composite. Their Marine reference is an excellent standard • to give identification ofeluting peaks)

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• • 40 Seafood Quality Control Laboratory Manual • PEROXIDE VALUE

Woyewoda, A.D. et al. 1986 Recommended Laboratory Methodsfor Assessment ofFish Quality, Can. Tech. Report ofFish. andAquatic Sci. No. 1448, Dept. ofFish. and Oceans, P.o. Box 550, Halifax, • Nova Scotia, B3J287 INTRODUCTION: Peroxide value is one ofthe most frequently used procedures for the examination oflipid oxidation. Peroxides form early in the oxidation stages,just subsequent to conjugateddienes. As the hydrogen is abstracted from the lipid's carbon backbone a site opens up for the addition ofan oxygen molecule (02), and a hydrogen molecule (H). Upon the addition ofthese three molecules a peroxide is formed. c-c=c-c-c=c-c -+ C-C-C=C-C=C-C -+ C-C-C=C-C=C-C • • 0 + o .OOH H (peroxide) Subsequent to peroxide development, the lipid undergoes more reactions which end in a breakage, on either side ofthe peroxide, ofthe lipid backbone made up ofcarbon molecules. Small fragments ofthe • lipid now become volatile (due to their small size) an contribute to the detectable rancid, oxidized odors.

REAGENTS:

Acetic acid I chloroform solution: Mix 300 ml ofglacial acetic acid and 200 ml • chloroform. Sodium thiosulfate: stock (0.1 N): Place 24.8 g Na2S203· 5 H20 into a 1000 ml volumetric flask and bring to volume with distilled water. working (0.01 N): Accurately pipet 10 ml of 0.1 N stock solution into a 100 ml volumetric flask, and bring to volume with distilled water. • Potassium iodide (0.904 M): Dissolve 15 g potassium iodide in distilled water, and bring to volume with additional distilled water in a 100 ml volumetric flask.

Saturated potassium iodide solution: To 20 ml ofrecently boiled distilled water, add KI until crystals remain undissolved. Store in an amber bottle at refrigerated temperature until use. Ifsolution is not colorless, prepare fresh prior to peroxide testing.

• Starch indicator solution: Dissolve 1 g ofstarch into 10 ml cold, distilled water and add to 100 ml boiling distilled water. Boil an additional 1 minute. Cool prior to use. Prepare fresh daily.

Standard potassium dichromate solution (0.1 N): Dissolve 0.490 g K2Cr207 (dried) with 80 ml distilled water. Mix for complete dispersion prior to bringing up to 100 ml with • distilled water, in a volumetric flask. Hydrochloric acid

PROCEDURE:

• Standardization of 0.1 N thiosulfate:

• Pipet 25 ml of standard potassium dichromate solution into a 125 ml Erlenmeyer flask.

• Add 5 ml concentrated HCl and 10 ml 0.904 M KI solution. Mix and let stand 5 minutes. • 41 Seafood Quality Control Laboratory Manual • • Add 100 ml distilled water and titrate with 0.1 N thiosulfate solution until yellow colorjust disappears.

• Add approximately 0.5 ml starch indicator with pasteur pipette. • • Continue titrating with 0.1 N thiosulfate, with constant stirring, until blue colorjust disappears. CALCULATION OF SODIUM THIOSULFATE NORMALITY:

Normality ofNa1S10J = 2.5/ volume of thiosulfate required for titration

• PEROXIDE VALUE DETERMINATION: • Place 2.5 - 5.0 g accurately weighed lipid into 250 ml iodine flask, and flush with nitrogen or other inert gas.

• Add 30 ml acetic acid I chlorofonn solution to dissolve lipid. • • Add I ml saturated KI solution and swirl gently for exactly 2 minutes. • Add 100 ml distilled water and approximately 1 ml starch solution.

• Titrate with 0.01 N sodium thiosulfate with vigorous stirring until blue color disappears. • •A blank titration should be perfonned for each Peroxide Value session.

CALCULATIONS:

Peroxide Value (POV) =(ml ofthiosulfate) (N of thiosulfate) (1000) I wgt oflipid in grams. •

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• • 42 Seafood Quality Control Laboratory Manual • CONJUGATED DIENES

Buege, J.A. andAust, S.D. 1978 In: Methods in Enzymology, Vol 52. p. 302 -310. Academic Press, New York

INTRODUCTION: The first steps in the breakdown or oxidation oflipid is the abstraction ofa • hydrogen molecule from the lipids carbon backbone. The abstraction takes place at one end ofa double bond and is termed the "initiation" step. Within micro time subsequent to the hydrogen's abstraction, the lipid reduces its overall energy by flopping the double bond to an adjacent carbon. In many instances this sets up a condition known as conjugation, where two double bonds are separated by only one single bond ( C-C=C-C-C=C-C ~ C-C-C=C-C=C-C). This conjugated system has a unique absorption at around 233 nm, which allows it to be monitored. This procedure allows an examination oflipid oxidation at a very early time in the process, and can detect changes in lipid quality before • other procedures can do so.

REAGENTS:

Iso-octane! ethanol solution: Mix together 10 ml iso-octane and 10 ml of 100% ethanol.

• PROCEDURE:

• Place 250 mg oflipid extracted by the method ofBligh and Dyer (1959), either by direct weight or by placing a known volume ofa known concentration oflipid iniso-octane:ethanol solution, and drying under nitrogen. • Add 5.0 ml ofiso-octane:ethanol solution to the lipid. • • Remove 1.0 ml ofthis solution and transfer to a 25 m1 volumetric flask. • Bring up to volume with only ethanol (100%). • Read absorbance in a quartz cuvet at 233 nm. • CALCULATIONS: Report values in Molar concentrations.:

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• • 43 Seafood Quality Control Laboratory Manual • AEROBIC PLATE COUNT (APC)

FDA Bacteriological Analytical Manual, 7th ed. 1992, page 17

REAGENTS: • Butterfield's phosphate reagent Plate count agar

PROCEDURE:

• Place 50 g ofa representative sample ofseafood into a sterile Waring blender. • • Add 450 ml ofsterile Butterfield's phosphate buffered dilution water and blend for 2 minutes on high. This is a 10-1 dilution. 2 • Prepare serial dilutions of10. , 10·3, and 10-4 ofseafood homogenate by adding 1 ml ofa dilution to 99 ml ofdiluent to obtain the next lowest dilution. (Avoid getting foam in sample volume). • Shake diluted sample 25 times through an arc 30 cm. in 7 seconds. • Immediately pipet 1.0 ml into separate sterile Petri dishes. • • Add 12 - 15 ml plate count agar (cooled to 44-46CC) to each Petri dish. •A control is made by placing 1.0 ml ofsample diluent that contains no sample homogenate and 12­ 15 ml agar. • Immediately mix diluted sample or control and agar by a swirling back and forth motion alternated with a rotational motion on a flat surface. • Let set on counter until agar solidifies. (Do not stackplates during either the pouring or solidification steps). • • Incubate Petri dishes inverted for 48 ± 2 hours at 35°C in an incubator.

CALCULAnONS:

Choose dilutions for Petri plates that contain between 25-250 counts per plate. Count the entire plate including those ofpinpoint size. Total colony forming units (CFU) and serial dilution should be • recorded.

Results are reported using only two significant figures. When rounding off, go to the next higher number ifthe digit to br rounded offis 5,6,7,8, or 9. Ifthe digit to be rounded offis 1,2,3, or 4, round down. For example, 10,600 becomes 11,000 and 10,400 becomes 10,000. • APC I g seafood = CFU x dilution Example: CFU = 231, dilution factor is 10-4 = 230 x 104 = 2,300,000 = 2.3 x 106 •

• • 44 Seafood Quality Control Laboratory Manual • SALMONELLA

FDA, Bacteriological Analytical Manual 1992, 7th edition, page 51

REAGENTS: • MacConkey's agar Lysine Iron agar Triple Sugar Iron Xylose Lysine Deoxycholate agar Bismuth Sulfate agar Hektoen Enteric agar Selenite Cystine broth • Tetrathionate broth Sterile Lactose broth

PROCEDURE:

• Aseptically place 25g ofrepresentative sample into a sterile Waring blender. • Add 225 ml ofsterile lactose broth and blend on high for 2 minutes. • • Place homogenate into a sterile screw top container, let set for I hour at room temperature with the top securely fastened. • Mix well and adjust pH to 6.8 ± 0.2 ifneeded with 0.5 MNaOH or HCl. • Add 2.25 ml ofsteamed (15 minutes) Triton X-IOO to initiate foaming. • Loosen screw top covers and incubate at 35°C for 24 ± 2 hours. • Tighten lid and gently shake mixture. • • Transfer 1 ml ofincubated homogenate to 10 ml oftetrathionate broth (IT), and 1 ml to 10 ml of selenite cystine broth (SC).. • Incubate for 24 ± 2 hours at 35°C. • Vortex® both broth tubes and streak 3mmloopfuls onto 1) bismuth sulfite agar, 2) xylose lysine deoxycholate agar, and Hektoen enteric agar. • Incubate plates for 24 ± 2 hours at 35°C. • Count and record number ofCFU for each plate • Hektoen enteric agar blue-green colonies. Bismuth sulfite agar appear dark and have a metallic sheen. Xylose lysine deoxycholate agar appear pink with or without dark centers. • Aseptically transfer 2 or more colonies suspected to be Salmonella to Triple Sugar Iron (TSI) slants and Lysine Iron Agar (LIA) slants. Use stab and streak methods. LIA slants must have deep butt to ensure an anaerobic environment. • • Incubate TSI agar at 35°C for 24 ± 2 hours, and LIA agar at 35°C for 48 ± 2 hours. Both agars should be incubated with cap loosely on In TSI agar Salmonella produces red slant and yellow butts (with or without the production ofagar blackening HzS). In LIA agar Salmonella produces purple butt. (consider yellow in butt to be negative reaction). • Streak representative colonies onto MacConkey's agar and incubate at 35°C for 24 ± 2 hours • • Examine plates for Salmonella, which appear as colorless and transparent colonies, with or without black centers. • Suspect colonies can further tested using biochemical and serological tests as described in FDA, BAM. • • 45 Seafood Quality Control Laboratory Manual • STAPHYLOCOCCUSAUREUS

FDA Bacteriological Analytical Manual, 1992 7th edition, page 161.

REAGENTS: • Butterfield's Reagent Baird-Parker agar Brain Heart Infusion broth Tyrpticase Soy agar Coagulase Plasma Hydrogen Peroxide solution Paraffin Oil • Phosphate-saline buffer Lysostaphin Toludine Blue Deoxyribonucleic acid agar

PROCEDURE:

• Place 50 g ofseafood into a sterile Waring blender. • • Aseptically add 450 ml ofsterile Butterfield's phosphate-buffered dilution water, and blend on high speed for 2 minutes. lbis is a 10-1 dilution. • For each dilution, place 0.3 - 0.4 ml ofhomogenate onto the surface ofsolidifiedBaird-Parker plates. • Spread homogenate over the plates surface using a sterile bent glass rod. • Place plate upright into an incubator for 1 hour at 35°C. • • After 1 hour, invert the plates an continue to incubate for a total of45-48 hours. • Colonies appear as circular (2-3rom dia.), smooth, convex and gray to black in color. (Other appearances may become evident ifpresent in a large population). • Select dilution with the lowest colony count and count and recordCFUs.

Conformation tests • Coagulase test • Pick suspect colonies and aseptically transfer to tubes continuing 0.2-0.3 ml ofBrain Heart Infusion broth, mix thoroughly. • Inoculate a solidified slant made oftrypticase soy agar. • Incubate tubes at 35°C for 18-24 hours. • Add 0.5 ml ofcoagulase plasma with EDTA to the culture and mix well. • Incubate at 35°C and examine for clot formation over a 6 hour period. • •A positive response for S.aureus is a very firm clot. Partial clotting requires further testing.

Catalase • Transfer a loopfull ofsuspect colonies to a clean glass plate. • Add a drop of hydrogen peroxide solution and examine for small bubbles, which is the indicator • for a positive reaction. Anaerobic glucose • Inoculate a sterile tube containing approximately 10 ml ofa fermentation medium containing glucose. • Cover over inoculated medium with a 25 rom thick layer ofparaffin oil. • Incubate at 37°C for 5 days. • The development ofa yellow color is an indicator ofa positive response.

• Lysostaphin • Select suspect colonies and aseptically transfer to a sterile tube containing 0.2 ml ofphosphate­ saline buffer and mix well. • • Add 0.1 ml oflysostaphin at a concentration of25J.1g1ml. 46 Seafood Quality Control Laboratory Manual • • Incubate tubes at 35°C for 2 hours or less. A positive response is indicated by a definite clearing of the turbidity.

Thermostable nuclease • Spread toludine blue-deoxyribonucleic acid agar on a microscope slide and let solidify. • Using a pasteur pipet tip cut and remove plugs ofthe agar. • • Heat sample to be tested (broth cultures) in boiling water bath for 15 minutes. • Add 0.1 ml to each well cut in the agar. • Incubate slides in a moist environment for 4 hours •A positive response is indicated by the development ofa pink ring around each well. • In all the ancillary tests described above S.aureus would give a positive response.

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• • 47 Seafood Quality Control Laboratory Manual • LISTERIA MONOCYTOGENES

FDA Bacteriological Analytical Manual 1992, 7th edition, page 141.

REAGENTS: • Sterile Enrichment broth Sodium Pyruvate (O.l%) Oxford Medium agar Lithium Chloride-Phnylethanol-Moxalactam agar Saline (O.85%) Hydrogen Peroxide solution • Sheeps Blood Agar (5%) PROCEDURE:

• Place 25 g ofa representative sample ofseafood into a sterile Waring blender. • Add 225 ml ofsterile enrichment broth and blend on high for 2 minutes. • Transfer to a sterile flask and incubate at 30°C for 2 days. • An enrichment step for stressed cells can be performed by the addition ofan additional enrichment • broth containing 0.1 % sodiwn pyruvate and incubating at 300C for 6 hours. Selective agents, if needed can be added at that time and additional incubation for a total of48 hours would then be performed. • At 24 and 48 hours transfer (streak) cultures from enrichment broth to 1) Oxford mediwn (OXA) and 2) lithiwn chloride-phenylethanol-moxalactam (LPM) agars. • Incubate for 24-48 hours at the following temperatures, OXA 35"C and LPM at 30°C. • • Examine using 45° angle light appear bluish on LPM agar and black with halos on OXA media.· Counting colonies on the OXA media is greatly assisted by illumination by Henry's apparatus.

Identification • Transfer suspect colonies offboth the OXA and LPM plates to sterile plates containingtrypticase soy agar with 0.6% yeast extract. • Incubate at 30°C for 24-48 hours. • • Colonies on this media appear blue-gray and should be selected and further tested

Mobility • Transfer a suspect colony oforganisms using sterile 0.85% saline and examine with a microscope for mobility. Listeria are mobile short rods.

Catalase • • Place suspect colonies onto a clean microscope slide and place a drop ofhydrogen peroxide solution onto the colonies. The development ofbubbles is a positive result.

Blood agar plates • Inoculate 5% sheep blood agar plates by deeply stabbing into media. Draw a grid on the plates bottom with between 20-25 spaces. L. monocytogenes produces clear zones around the inoculation • sight. The use ofserological tests can also be performed with many companies supplying test kits., such as Gen-Probe™(San Diego, CAl or ElISA DNA probe kits (GeneTrak, Framingham, MA).

There is no official method to enwnerate Listeria plate counts but it is advised to do so at a step prior to any enrichment step. • • 48 Seafood Quality Control Laboratory Manual • ESCHERICHIA COLI

FDA Bacteriological Analytical Manual 1992, 7th edition, page 27.

INTRODUCTION: E.coli is used as a sanitary index for cleanliness, or as an indicator organism for • fecal contamination. The bacteria, as are all coliforms, is Gram-negative, and rod shaped. They are identified by being gas producers from a variety ofsugars and fermentative oflactose.

REAGENTS: Butterfield's phosphate dilution water: Lauryl tryptose broth (LTB) Brilliant green lactose broth (BGLB) • Violet red bile agar (VRBA) Kovac's reagent Methyl red Voges-Proskauer reagent (MR-VP) Koser's citrate broth a-naphthol solution Potassium hydroxide: Place 400 g ofpotassium hydroxide into approximately 700 ml of distilled water in a fume hood and swirl until soluble. Let cool and top offto 1000 ml with • distilled water in a volumetric flask

PROCEDURE:

Presumptive test • Place 25 g ofrepresentative seafood into a sterile Waring blender. • Add 225 ml ofsterile Butterfield's phosphate-buffered dilution water, and blend on high speed for • 2 minutes. This is a 10'\ dilution. Subsequent dilutions can be made by placing 10 ml of homogenate into 90 ml ofsterile dilution water. • Shake all dilutions 25 times for 7 seconds through an arc of30 cm. • Aseptically transfer 1 ml portions ofsample for 3 consecutive dilutions into 20 ml ofsterile lauryl tryptose broth (LST) in separate tube. • Incubate tubes for 48 ± 2 hours at 35°C • • Examine tubes for gas production or effervescence when tubes are slightly shaken. • Detection ofgas indicates a positive presumptive test.

Confirmed test • Aseptically transfer a loopful ofsuspension from positive samples in the presumptive test to a tube filled with approximately 15 ml of Brilliant green lactose bile broth (BGLB). • Incubate the suspension for 48 ± 2 hours at 35°C. • • Examine for gas production, which indicates a positive response. • Record the positive responses for the 3 consecutive dilutions and calculate the number ofcoliform per g ofseafood by using the most probable number technique.

Coliform group • Prepare a homogenate (10'\) as described above. • Pour 10 ml ofViolet red bile agar (VRBA) tempered to 48°C into Petri dishes. • • Add 1 ml ofhomogenate and swirl plates on a flat surface, let solidify. • After solidification, add 5 ml addition VRBA evenly on the surface (overlay) and let solidify. • Invert plates and incubate for 18-24 hours at 35°C. • Count purple-red colonies 0.5 mm or greater surrounded by a clear zone. • This method has also been commercialized as Petrifilm® (see Appendix) • CALCULATIONS: coliform I g seafood = CFU x dilution factor

Confirmation for E.coli • 49 Seafood Quality Control Laboratory Manual • To all suspect Gram-negative short rods or cocci perform the following: Indole • Transfer suspect colonies aseptically into tubes ofsterile tryptone broth and incubate at 35°C for 24 ± 2 hours. • Add 0.2-0.3 ml ofKovac's reagent. A positive response is a red color that appears in the upper • layer.

Voges-Proskauer (VP) reactive • Inoculate MR-VP tubes asceptically with suspect colonies and incubate at 3SOC for 48 ± 2 hours. • Transfer 1 ml ofincubated broth to a clean test tube. • Add 0.6 ml ofa-naphtol and 0.2 ml of40% potassium hydroxide, then mix. • Add a few crystals ofsolid creatine. • • Mix and let set at room temperature for 2 hours. •A positive response is indicated by the development ofa pink color.

Methyl-red reactive • Incubate inoculated MR-VP broth tubes for 96 ± 2 hours at 35°C. • Add 5 drops ofmethyl red solution. A positive reaction is detected by the development ofa red • color, whereas, a yellow color indicates a negative response. Citrate • Inoculate a tube ofKoser's citrate broth with suspect colonies (lightly). • Incubate at 35°C for 96 ± 2 hours. •A positive response is indicated by the development ofa distinct turbidity.

• Lactose gas • Inoculate suspect colonies into a sterile tube of Lauryl tryptose broth. • Incubate at 35°C for 48 ± 2 hours. •A positive response is detected by the development ofeffervescence after slight agitation. • E. coli is confirmed ifthe following is found:

Indole + Indole Voges-Proskauer + Voges-Proskauer + Methyl-red Methyl-red Citrate Citrate Lactose gas + Lactose gas + • Gram negative + Gram negative +

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• • r • Petrifilm™ 3IVI

(English) Aerobic Count Plates To be used for total aerobic bacteria enumeration.

Description: • The Petrifilm Aerobic Count plate is a reliable, ready made medium system for enumerating total aerobic bacteria populations. (1.2.3,4). Petrifilm Aerobic Count plates contain Standard Methods nutrients, a cold water soluble gelling agen~ and a . tetrazolium indicator dye which facilitates colony enumeration. Warning: Petrifilm Aerobic Count plates are not intended to be used for in vitro diagnostic use. Storage and disposal: Store sealed Petrifilm Aerobic Count plate foil pouches at or below 46°F (8°C). After • opening, return unused plates to foil pouch. Seal foil pouch by folding and taping the open end. Store resealed foil pouches in a cool dry place. Use plates within one month after opening. Exposure of Petrifilm Aerobic Count plates to temperatures and/or humidities above 75°F (24°C) and 50% RH can affect the performance of the plates. Resealed foil pouches may be stored in freezer to protect plates from high temperatures and/or humidities. Do not use plates that show brown discoloration. After use, Petrifilm Aerobic Count plates will contain viable bacteria. Handle and discard appropriately. • Directions for use: 1. Place the Petrifilm Aerobic Count plate on a flat surface. 2. Lift top film. Hold pipette perpendicular to the plate and carefully dispense 1 ml of sample onto the center of bottom film. 3. Release top film down onto sample. 4. Distribute sample evenly using a gentle downward pressure on the center of the plastic spreader (recessed side down). Do not slide the spreader across the film. • Remove spreader and leave plate undisturbed for one minute to permit solidification of the gel. 5. Incubate plates in a horizontal position. with the clear side up in stacks not exceeding 20 plates. Follow current total plate count standards for incubation temperature. Temperatures above 37°C are not recommended. Incubate plates 48 ± 3 hr. 6. Petrifilm Aerobic Count plates can be counted on a standard colony counter. The reduction of the tetrazolium indicator dye will cause the colonies to become red. All • red dots regardless of size or intensity should be counted as colonies. The circular growth area is approximately 20 cm2• Estimates can be made on plates containing greater than- 250 colonies by counting a representative number of squares and mUltiplying by the appropriate number to obtain an estimated count for the total 20 cm2 growth area. The presence of very high concentrations of colonies on the plates will cause the entire growth area to become red or pink in color, record results as "too numerous to count" (TNTC). Occasionally, on overcrowded plates. the center may lack visible colonies but many small colonies will be seen on the edges. When thiS occurs, record results as TNTC. Some organisms can liquefy the gel, allowing them to • spread out and obscure the presence of other colonies. If a liquifier interferes with counting, an estimated count should be made by counting the unaffected areas. 7. To isolate colonies for further identification, lift the top film and pick the colony from the gel. References 1. Ginn, R.E., V.S. Packard, and T.L. Fox. 1986. Enumeration of Total Bacteria and Coliforms in Milk by Dry Rehydratable Film Methods: Collaborative Study. J. Assoc. Off. Anal. Chem. 69(3):527-531 • 2. McAllister, J.8., M.S. Ramos. and T.L. Fox. 1987. Evaluation of the 3M Dry Medium Culture Plate (Petrifilm'· SMJ Method for Enumerating Bacteria in Processed Fluid Milk Samples. Dairy and Food Sanitation 7(12):632-635. 3. McGoldrick, K.F., T.L. Fox, and J.S. McAllister. 1986. Evaluation of a Dry Medium for Detecting Contamination on Surfaces. Food Technology 40(4J:77-80. 4. Smith, L.B., T.L. Fox, and F.F. Busta. 1985. Comparison of a Dry Medium Culture Plate (Petrifilm 8M Plates) Method to the Aerobic Plate Count Method for Enumeration of Mesophilic Aerobic Colony-Forming Units in Fresh Ground Beef. J. Food Prot • 48(12J:1044-45. Instructions for specific dairy and food product applications and environmental surface sampling procedures are available upon request by calling your local Petrifilm Account Representative or Petrifilm Technical Services: (800) 328-6553 outside of Minnesota; (612) 736-1258 inside of Minnesota. • 3M Fetrifilm™ Aerobic Count Plates Reminders for Use • Storage

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-Refrigerate unopened packages To seal opened package, fold end Keep resealed package at gO°F I of 3MTM Petrifilm plates. Use 2 over and tape shut. 3 (::;21 0c), ::;50%RH. Do not • before expiration date on package. refrigerate opened packages. Use Petrifilm plates within one Sample Preparation month after opening. •

• Prepare a 1:10 or greater dilution of Add appropriate quantity of diluent. Blend or homogenize sample per 4 food sample. Weigh or pipette food 5 These include Standard Methods 6 current procedure. product into Whirl-Pac" bag, phosphate buffer, 0.1 % peptone stomacher bag, dilution bottle, or water, distilled water, phosphate other appropriate sterile container. buffered saline, and Butterfield's buffer. Do not use buffers containing sodium citrate or thiosulfate. • Inoculation

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Place Petrifilm plate on flat With pipette perpendicular to Release top film; allow it to DROP. 7 surface. Lift top film. 8 Petrifilm plate, place 1 ml of sample 9 Do NOT roll top film down. • onto center of bottom film.

• With RIDGE side down, place GENTLY apply pressure on spreader Lift spreader. Wait one minute for 10 spreader on top film over inoculum. 11 to distribute inoculum over circular 12 gel to solidify. area. Do not twist or slide the spreader. • Continued - over II • r

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Incubate Petrifilm plates with the Read Petrifilm plates ona standard .13 clear side up in stacks of 20 or 14 Quebec-type colony counter or less at a temperature of 32-35°C other magnified light source. Refer (90-95°F) for 48 ± 2 hours. to Guide to Interpretation when reading results. •

• Additional Comments Steps 9 and 10 are unique to Petrifilm Aerobic Count, plates.

Note: Remember to inoculate and spread each Petrifilm plate before going on to the next.

Questions? Call 1-800-328-6553 for technical service.

To order Petrifilm plates call 1-800-328-1671. • For general information on Petrifilm plates, call 1-800-228-3957. For more information on any 3M product, call 1-800-362-3456.

For information outside the United States, contact 3M Microbiology Products:

Canada, call: Ontario and Western Provinces 416-443-7800. • Quebec and Eastern Provinces 514-631-7600. Europe, FAX# 33-1-49-65-5100.

Mexico, call 52-5-626-0400.

Puerto Rico and Caribbean area call 809-750-3000. • All other locations outside the U.S., FAX# 612-737-1994. References 1. JAOAC. 1989 72 (2): 312-318. 2 JAOAC. 1990. 73 (2): 242-248.

• 3IVI Microbiology Products 3M Canada, Inc. 3M Health Care P.O. Box 5757 R('cn:led Paper Petrifilm is a trademark of 3M. 1'''l'~C01/Sllm('r. Whirl-Pac is a trademark 01 Nasca 1,M Center Bldg. 275-4E-O I London. Ontario N6A4TI -/O,;i- 10(/; p(I.\"J-nmSIIIJJ('I" Printed in U.S.A St. Paul. MN 55144-1000 Canada ©3M 1993 • 1-800-563-2921 70-2008-4763-3 (631)11 .>J .., .,,;,.,. .... "J;F~·

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Count = 0 Count = 16 It is easy to interpret the Petrifilm Aerobic Count plate. Figure 3 shows a Petrifilm Aerobic Count plate with a Figure 2 shows a Petrifilm Aerobic Count plate few bacterial colonies. A red indicator dye in the plate • without colonies. colors the colonies. Count all red colonies regardless of sizes or color intensities. Use a standard Quebec-type counter to read the Petrifilm plate.

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Count = 143 Estimated count = 420 As with an agar pour plate, the preferable counting range When colonies number more than 250 as in figure 5, on a Petrifilm Aerobic Count plate is 25-250 colonies. estimate the count. Determine the average number of • See figure 4. colonies in one square (l cm2) and multiply it by 20 to obtain the total count per plate. The inoculated area on a Petrifilm Aerobic Count plate is approximately 20 cm2.

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Count =TNTC Count =TNTC Figure 6 shows a Petrifilm Aerobic Count plate with With very high counts, the entire growth area may tum colonies that are too numerous to count (TNTC). pink, as shown in figure 7. You might observe individual • colonies only at the edge of the growth area, Record this as a TNTC result.

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8 9 Count =TNTC Count =TNTC Occasionally, distribution of colonies appears uneven as The colonies on the Petrifilm Aerobic Count plate in shown in figure 8. This is also an indication of a TNTC figure 9 appear countable at first glance. However, when • result. In fact, the distribution is even. you look closely at the edges of the growth area, you can see a high concentration of colonies. Record this as a TNTC result. See figure 9.

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Estimated count =160 Count = 83 A few species of bacteria liquify the gel in the Petrifilm Because colonies on Petrifilm Aerobic Count plates are Aerobic Count plate, as shown in figure 10. When this red, you can distinguish them from opaque food particles • occurs, determine the average count in a few unaffected that cause confusion with agar pour plates: See figure II. squares and then estimate the total count. Do not count red spots within the Iiquified area. • For technical assistance. call 1-800-328-6553. For information outside the United States, contact To order Petrifilm plates, call 1-800-328-1671. 3M Microbiology Products: For general information on Petrifilm plates, call 1-800-228-3957. Canada, call 1-800-563-2921 or Ontario and Western Provinces 416-443-7800. For more information on any 3M product. call 1-800-362-3456 Quebec and Eastern Provinces 514-631-7600. Europe, FAXII 33-1-49-65-5100. • Mexico, call 52-5-626-0400. Puerto Rico and the Caribbean area call 809-750-3000. All other locations outside the U.S .. FAX 612-737-1994. •

3NI Microbiolo!G' Products 3M Canada, Inc. • 3M Center. Buildinc 275-4E-O\ P.O. Box 5757 Recycled Paper SI. Paul. MN 55144-1000 London. Ontario N6A4T\ Pelrllrlm IS a trademark or ]M USA Canada Prrnted In USA \-ROO-nR-3957 1- 800-563-292\ ©3M '993 70-2008'4572,8 r 123 81" • l'etrlfIlml ~I E. coli Count Plates (English)

• To be used for the enumeration of Escherichia coli and coliforms. Description: The Petrifilm E. coli Count plate is it reliable. sample-ready medium system for enumerating Escherichia coli and coliforms. Petri film E. coli Count plates contain \'iolet red bile nutrients. a cold water soluble gelling agent. a glucuronidase indicator to identify E. coli.. and a tetra­ zolium indicator to enhance the visualization of other gram negative I non-E. coli) bacteria. Colifonns fennent the lactose in the medium to produce gas. This gas is trdpped around the • colifonn colony and allows the differentiation of coliform bacteria from other gram negati\e bacteria. In addition. glucuronidase. produced by most E. coli* will react with the glucuronidase indicator in the medium to produce a blue precipitate around the colony allowing visual identification of E. coli. Warning: Petri film E. coli Count plates are not intended for in vitro diagnostic use. Storage and disposal: Store sealed Petrifilm E. coli Count plate foil pouches at or below 46 cF (8°C). After opening. return unused plates to foil pouch. Seal foil pouch by folding and taping the open end. Store • resealed foil pouch in a cool dry place. Use plates within one month after opening. Exposure of Petrifilm E. coli Count plates to temperatures and/or humidities above i5'F (24 cC) and 50'7e RH can affect the perfonnance of the plates. Do not use plates that show orange or brown discoloration. After use, Petrifilm E. coli Count plates will contain viable bacteria. Follow current industry standards for disposal. Directions for use: • I. Place the Petrifilm E. coli Count plate on a flat surface (see figure I). 2. Lift top film and dispense I mJ of sample onto the center of the bOllom film (see figure 2). 3. Slowly roll the top film down onto the sample to prevent the entrapment of air bubbles (see figure 3). 4. Distribute sample evenly within the circular well using a gentle downward pressure on the center of the plastic spreader (flat side down) (see figure 4). Do not slide spreader across the film. Remove spreader and leave plate undisturbed for one minute to pennit solidification of the gel. 5. Incubate plates in a horizontal position with the clear side up in stacks not exceeding 20 • plates. F,ollow current industry standards for incubation temperature. Incubate plates 24:t 2 hr and examine for colifonn and E. coli growth. Some E. coli colonies require additional time to fonn the blue precipitate. Reincubate plates an additional 24 :t 2 hr. to detect any additional E. coli growth. oa. Slowly count Petrifilm E. coli Count plates on a standard colony counter. E coli colonies \\ ill appear blue (see figure 6a). Blue colonies associated with gas are confirmed E. coli. . Other colifonn colonies will be red and associated with eas (bubbles \\ ill be seen in the medium). Colonies not associated with gas (with it dista~ce greater than one colony diameter • from gas bubble) are not counted as coli forms. The total coliform count consists of the red colonies with gas and blue colonies with gas at 24 hours. Do not count colonies on the white foam dam since they are removed from the selective influence of the medium. The circular growth area is 20cm~. Estimates can be made on plates containing greater than 150 colonies by counting a representative number of squares and multiplying by the appropriate number to obtain an estimated count for the total 20 cm~ growth area. 6b. High concentrations of E. coli will cause the growth area to tum a bluish color (see figure 6b I J, while high concentrations of colifonns (non-E. coli) will cause the growth area to tum a • dark reddish color (see figure 6b2). When this occurs. further dilution of the sample is required to obtain a more accurate count. 7. To isolate colonies for funher identification, lift the top film and pick the colony from the gel (see figure 7). *Some E. coli. such as E. coli OI57:H7, are glucuronidase negative and will not produce the blue precipitate. Additional infonnation on Petrifilm E. coli Count plates can be obtained from your local Petrifilm sales representative or Petrifilm Technical Service: 800-328-6553 (outside of Minnesota): • 6121736-1258 (inside Minnesota). Outside of the United States. contact the local 3M subsidiary. In Canada. contact 3M Canada. Inc.. P.O. Box 5757. London. Ontario. 1"6,\ 4TI: 1-800-268-9696. 3NI

Made in U.S.A. for Petrlfilm IS a tradema'< of 3M 3M Health Care Printed In USA • St. Paul. MN 55144-1000 ©3M 1993 34-7034-9221-4

• BESTAV/J,ILABLE COpy I • IetrifIlm™ Colifonn & E. coli Count Plates Reminders for Use

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Refrigerate unopened packages. Use To seal opened package, fold end Keep resealed package at :::;70°F • 1 before expiration date on package. 2 over and tape shut. 3 (:::;21°C), ::;SO%RH. Do not refrigerate opened packages. Use Petrifilm plates within one month after opening. •

• Prepare a 1:10 or greater dilution of Add appropriate quantity of diluent. Blend or homogenize sample per 4 food product." Weigh or pipette food 5 These include Standard Methods 6 current procedure. product into Whirl-Pac' bag, phosphate buffer, 0.1 % peptone stomacher bag, dilution bottle or water, distilled water, phosphate " If greater sensitivity is required other appropriate sterile container. buffered saline, and Butterfield's with dairy or juice products please buffer. Do not use buffers containing refer to Petrifilm Dairy & Juice • sodium citrate or thiosulfate. Products sheet.

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Place Petrifilm plate on flat With pipette perpendicular to Carefully roll top film down to 7 surface. Lift top film. 8 Petrifilm plate, place 1 ml of sample 9 avoid entrapping air bubbles. • onto center of bottom film. Do not let top film drop.

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With flat side down, place Gently apply pressure on spreader to Lift spreader. Wait one minute for 10 spreader on top film over inoculum. 11 distribute inoculum over circular area. 12 gel to solidify. • Do not twist or slide the spreader. Continued - over • In~on •

Incubate Petrifilm plates with the Read Petrifilm plates on a standard • 13 clear side up in stacks of 20 or less 14 Quebec-type colony counter or at a temperature of 32-35°C (90­ other magnified light source. 95°F) for 24-48 hours. Refer to Guides to Interpretation when reading results. •

•

• Additi~ments

.No.ferRemember.:I01QocOlateah~l~pr~W£n~~ch'Retfifilm platebe'fR~e g~oingqt1-!~lhe rie~, • Steps 9 and 10 are unique to Petrifilm Coliform Count and E. coli Count plates. QLlestion~?·CaI11l§,Qm~?if65!?~~&~~n!~i§irYl~S

", .'~'-' ~T ,' ""0'''~!f;P:;~'''1i'<''':;~';r'-,' """'~~·~:;'~i:\..'t1p,.:'%~At;;~ Td order·Petrifilmplates'call''1 ;:8QO:::328~1671r

Outside the United States, contact 3M Microbiology Products at FAX number 1-612-737-1994. •

References 1. J. Food. Prot. 1990.53 (2): 145-150. 2. JAOAC. 1991.74(4): 635-648.

• 70-2008-4764-1 (32.5);i Petrifilm is a trademark of 3M. ©3M,1992 Microbiology Products 3M Health Care 3M Center Bldg. 275-4E-01 3IVI • 81. Paul, MN 55144-1000 .;f"In~" "t ";i~llfitJ'~~!~~t .",{,? erpre~ 10h'{~s:!pr, e~ .' Petrifilm • E. coli Count Plate

·This guide familiarizes you with results on Petrifilm brand E. coli Count plates. For more information, call 1-800-328-6553, • or contact your 3M Microbiology Products sales representative.

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E. coli count =50; coliform count =91 E. coli count = 3; coliform count = 6 • It IS easy to count L. (oli colonies on Petrifilm E. coli Circle, I and 2 in figure I also ,how how hubble Count plates. /-", (oli produces glucuronidase which pattern, can vary. SOllletime, gas di'fupts the colony ,0 reacts with an indicator dye in the plate to form ;1 blue th;lt the colony "outlines" the bubble, as in figure 2. prt:'cipitate around the colony. Most t:. coli also produce l'lrcle I. g;\S ",hil'h the top film traps, Count blue colonic, a,soclated with gas bubbles as confirmed F (oli. :\rtiLll'l bubhles may result from improper inoculatioll See figure I. circle I. of the Petrifilm E. coli plate. They are irregularly shaped • alld are not as',ociated with a red colony. Sec figure 2, Non·f (oIl colifmnh form red colonies and are l'ircle 2.

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E. coli count =0 E. coli count = 17 Notice the change in gel color in figures 3 through 8. • As the E, coli count increases, the color of the gel turns to a purple-blue.

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E. coli count = 114 Estimated E. coli count = 500 The prcferahle counting range is 15-150 colonies. When colonies number more than 150. estimate the count. See figure 5. Determine the average number of colonies in one square (I cm") and multiply it by 20 to obtain the total count per • plate The growth area on a Petri film E. coli plate is 20 cm2 See figure 6.

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E. coli count =TNTC E. coli count =TNTC Petrifilm E. coli plates with colonies that are too [n figure 8, the count is so high that individual colonies numerous to count (TNTC) have one or more of the and gas bubbles do not show. The purple-blue gel color • following features: I) many small colonies, 2) many gas indicates that the result is TNTC. huhhles, 'lilt! 1) purple-hlue gel color. See figure 7.

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E. coli count =TNTC E. coli count =2 The Petri film E. coli plate in figure 9 has two features Figure 1() shows a Petrifilm E. coli plate with a few indicating that the result is TNTC: I) the purple-blue E, coli colonies and a high number of non-coliform, gel color. and 2) many gas bubbles. gram-negative colonies. When high numbers of • organisms such as Pseudomonas are present, the foam ane! gel turn yellow, as in figure 10. • •

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E. coli count =1 Food particles are often irregularly shaped and are not associated with gas bubbles. See figure 11, circle 1. • Blue colonies without gas mayor may not be E. coli; if necessary, confirm these colonies. See figure 11, circle 2.

• For technical assistance, call 1-800-328-6553. For information outside the United States, contact To order Petrifilm plates, call 1-800-328-1671. 3M Microbiology Products: For general information on Petrifilm plates, call 1-800-228-3957. Canada, call 1-800-563-2921 or Ontario and Western Provinces 416-443-7800. For more information on any 3M product, call 1-800-362-3456. Quebec and Eastern Provinces 514-631-7600. Europe, FAX# 33-1-49-65-5100. Mexico, call 52-5-728-2202, FAX# 52-5 -728-2299. • Puerto Rico and the Caribbean area call 809 -750- 3000. All other locations outside the U.S., FAX# 612-737-1994.

•

Microbiology Products 3M Canada, Inc. 3M Center, Building 275-4E-01 P.O. Box 5757 Recycled Paper • S1. Paul, MN 55144-1000 London, Ontario N6A4T1 Petrililm IS a trademark of 3M USA Canada Pnnled In U.S.A 1-800-228-3957 1-800-563-2921 ©3M 1993 70·2008·4574·4 (941 Iii • / ~> •.Interpret3tion'Guide~': ,. PetrifilDi1;._•. • Coliform Count Plate

This guidefamiliarizes you with results on Petrifilm brand Colifonn Count plates. For more infonnation, call 1·800·328·6553, • or contact your 3M Microbiology Products sales representative.

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Coliform count = 73 Coliform count = S. • It I~ ea~y to count coliform colonies on Petrifilm Arllfact huhhle~ may result from imrropcr inoculatilln Coliform Count plates. A red indicator dye in the pl;lle of the Petrifilm Coliform Count plate. They are colors all colonie~. and the top film traps gas produced irregularly shared and not associated with a red colony. h~ the coliform~. See figure 1. cirl'il' 2.

Collform.~ produce red colonie~ which are a~~oci;lled Do not count l'olonies whldl appear on the fO;II11 halTll'r with gas huhhle~. See figure I. circles 1.1 and 3. Non­ hecause they arc removed from the selective influence • coliforms produce red colonies which are not associated or till' medium. with gas huhhles. Sec figure 2. circle I.

Cirl'le~ 1.2 and .' in fi!!.ure I also show how huhhle P;IIIlTn~ can \'ary. Som~times ga~ disrupts the colon~ '0 that the colony "outlines" the huhhle as in circle ". • • •

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Coliform count = 0 Coliform count = 9 Notice the change in gel color in figures 3 through ~. As the coliform count increases, the color of the gel deepens • from a light pink to a purple-red.

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Coliform count = 79 Estimated coliform count = 220 ,'\s with viokt red hile agar plales. the preferahle counting When colonies numher more than I:">0. estimate the range on Petnfilm Coliform Counl plates is I)-]:">() coun1. Dctclll1ine the average number of colonies in one colonies, Sec figure :,,>, square (I cm~) and multiply It hy 20 to ohtain the tOLl] • Ulunt ]1n plate, The growth area on a Petrifilm Colifolln Count plale j's approximately 20 cme. See figure fl. • •

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Coliform count = TNTC Coliform count = TNTC Petrifilm Colifolln Count plates with colonies that arc In figure 8, the count is so high that individual colonies too numerous to count (TNTC) have one or more of the and gas buhbles do not show. A deepening of the gel following features: I) many small colonies. 2) many gas color indicates that the result is TNTC. • huhbles. and 3) deepening of the gel color, See figure 7.

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Coliform count = TNTC ('oliform count =4 The Petrifilm Colifonn COUllt plate in figure l) ha\ two Fi~ure I() stH1\\S a Petrifilm Coliform Count pL.lte with a rcaturc\ indicating TNTC colonle\: I) deepenini! of k\\ coliform colonies and a high numher of non­ the gel color. and 2) many gas huhhles. l·ol,fornl. gram-negative colonies. When high numhers of • or~anlsills such as Pseudomo!1as are present. the foam and ge I turn ye Ilow. as in figure In. • •

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Coliform count = 5 Coliform count = 2

Figure 11 shows a Petri film Coliform Count plate with Food panicles often are irregularl} shaped and arc IlOt coliforrn colonies in an icc cream sample, The colonies associated with gas bubbles. See figure 12. • and the gel have a slightly different appearance.

• hll' tl:chnical aSsistance, call 1-800-328-6553. For information outside the United States, contact 'I'll nrdl:r Pl:trilillll platl:s. call 1-800-.'128-1671. 3M MicrohioloJZY Products: h,r gl:lll:ral inforlllation on Petrltillll plates. call 1-800-228-.'1957. Canada.l'alll-XOO·:;63·292Ior Ontario and Westl:rn Provinces 416-..\43 -7X()() h II' Illore IIIform at Ion on any lM product. call 1- 800- 362· .1,..\:;6 Quebec and Eastern Provinces 514 -6ll-7fl()O, Europe, FAXII.\.\-1-4lJ-65-:;IOn • Mexico, call :;2·:;-62()-0400 Pucrto Rico and the Caribbean arl:a call XOY -7:;0- .\0(10 All othl:r \oL'atlolh IHlhiJe the liS, f'AXII 612·7.\7-1')'14 •

3M ,\1il'rohiulug,\ Products ,'\1 Canada. Inl'. • .'M Cl:nlcr. HuildllH! 27'i·4r.·OI PO Hox :; 7'i 7 Sl. I';IUJ. MN 'i'i 144' 1000 I.lllldoll, Onlarlo NhA4T I LISA Canada I· XOO· 22X .N:;7 I. xon -'i6.\ . 2LJ 2 I "'W''I'', 7020084573,6 16481" • Rt:-STAVAUAFLE COpy ~trifl1mTM Plates Environmental Monitoring Procedures • Air & Surface Sampling

Petrifilm plates are convenient, reliable methods for detecting environmental microbial contamination. The construction of Petrifilm plates allows the product to be used for air sampling, as well as direct contact or swab contact monitoring procedures.

A number of diluents can be used with Petrifilm plates. These include Standard Methods buffer, Butterfield's buffer, 0.1 % peptone water and letheen broth. Do not use diluents containing thiosulfate or sodium citrate. If sanitizers may be present, use letheen • broth for both the direct contact and swab contact methods. Letheen broth effectively neutralizes halogen, quaternary ammonium, and acid sanitizers.

Air Sampling Method

• Petrifilm plates can be positioned either horizontally or vertically for environmental air sampling.

Before using Petrifilm plates for air testing, hydrate plates with 1 ml of appropriate diluent (see above). Allow hydrated plates to remain closed for a minimum of 1 hour before use. It is not necessary for Petrifilm plate gel to be fully formed . •

For horizontal positioning use a Without touching circular growth Double stick tape can be used with or • Petrifilm plate clip in combination with area, lift top film portion of hydrated without clip for vertical positioning of double stick removable poster tape. plate and.'peel back until outer portion Petrifilm plates for air sampling. Position hinged edge of Petrifilm plate of film adheres to the tape. Make sure into clip. Apply a small piece of top film lies flat across clip. Expose double stick tape to each end of the Petrifilm plate to air for no longer than clip handle. 15 minutes. Remove film; incubate and • enumerate as directed. Swab Contact Method The procedure described below is derived from the methodology recommended by the American Public Health Association. Other swabbing techniques or swab rinse volumes may be substituted. However, the rinse solution used must be compatible with Petrifilm plates (see above for recommended diluents). Cotton swabs are recommended over the use of calcium alginate swabs. • If calcium alginate swabs must be used, then letheen broth containing 1% sodium glycerQphosphate should be used as the rinse solution and solubilizing agent respectively.

•

Dispense rinse solution into small Hold swab handle to make a 30° angle After the area has been swabbed, break screw-capped vials that will contain with the surface. Rub the swab slowly off the swab head into the rinse solution 5 mls of solution after autoclaving. and thoroughly over desired surface and replace cap. Shake vial containing Moisten the swab head and press out area. Rub the swab three times over swab head vigorously for 10 seconds. • excess solution against the interior wall this surface, reversing direction Inoculate Petrifilm plates with 1 ml of with a rotating motion. between successive strokes. solution. Incubate and enumerate as directed in package inserts. Refer to Guides to Interpretation when • reading results. • Direct Contact Method Storage Allow a minimum of 30 minutes at room Store hydrated Petrifilm plates in temperature for gel to solidify on Petrifilm pouch or plastic bag sealed with tape. Aerobic Count plates, and one to two Protect plates from light (foil pouch hours for gel to solidify on Petrifilm may be used) and refrigerate at 2-8°C Coliform and E.coli Count plates, before (36°-46°F). using plates for surface sampling. Hydrated Petrifilm Aerobic Count plates In the case of Petrifilm Yeast and Mold may be refrigerated up to 14 days. Count plates hydrate only with Other hydrated Petrifilm plates may be letheen broth for direct contact surface refrigerated up to 7 days. Hydrate Petrifilm plates according to monitoring. Place letheen inoculated instructions in package insert with plates into sealable bag and Incubate 1 ml oLappropriate sterile diluent at 30-37°C (86°-99°F) for 24 hours. • (see other side). After incubation, store sealed bag of plates in refrigerator for a minimum of 4 hours to allow gel to solidify. Petrifilm plates hydrated with letheen have a mottled appearance. • Surface Sampling Procedure

•

When gel is solid, carefully lift top film Allow the circular gel portion of the top Lift film from surface and rejoin the top portion of hydrated plate. Avoid film to contact the surface being tested. and bottom sheets of Petrifilm plates. touching circular growth area. Gel Rub fingers over the outer film side of Incubate and enumerate as directed in will adhere to top film. the gelled area to ensure good contact package inserts. Refer to Guides to with surface. As with any surface Interpretation when reading results. • sampling method the best microbial practice is to wipe surface after contact.

Air Sampling Method Results Swab Contact Method Results Direct Contact Method Results

2 Result is count/40cm2 for: Petrifilm plate count Result is count/20cm for: •• Petrifilm Aerobic Count plates x • Petrifilm Aerobic Count plates • Petrifilm Coliform Count plates volume of diluent (i.e. number of mls.) • Petrifilm Coliform Count plates • Petrifilm E.coli Count plates • Petrifilm E.coli Count plates total count I area sampled Result is count/60cm 2 for: Result is count/30cm2 for: • Petrifilm Yeast & Mold Count plates • Petrifilm Yeast & Mold Count plates

•• For technical assistance please call: 1-800-328-6553.

• To order Petrifilm plates or complimentary tape and clip, available in the U.S., call 1-800-328-1671 , or contact your regional sales representative.

• For information outside the United States, contact 3M Microbiology Products: Canada: Ontario and Western Provinces 416-449-8010; Quebec and Eastern Provinces 514-631-7600. Europe: FAX# 33-1-49-65-5100. Mexico: call 52-5-626-0400. All other locations outside the U.S., FAX# 612-737-1994.

• 70-2008-2412-9 (13.5);; Petrifilm is a trademark of 3M. ©3M,1993 Microbiology Products 3M Health Care 3M Center Bldg. 275-4E-01 3IVI • St. Paul, MN 55144-1000